WO2016090347A1 - Identification de vsig8 en tant que récepteur putatif de vista et son utilisation pour produire des modulateurs de vista/vsig8 - Google Patents

Identification de vsig8 en tant que récepteur putatif de vista et son utilisation pour produire des modulateurs de vista/vsig8 Download PDF

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WO2016090347A1
WO2016090347A1 PCT/US2015/064146 US2015064146W WO2016090347A1 WO 2016090347 A1 WO2016090347 A1 WO 2016090347A1 US 2015064146 W US2015064146 W US 2015064146W WO 2016090347 A1 WO2016090347 A1 WO 2016090347A1
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vsig8
cell
antibody
cells
vista
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PCT/US2015/064146
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English (en)
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Michael Molloy
Yalin GUO
Jay ROTHSTEIN
Michael Rosenzweig
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Immunext, Inc.
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Priority to JP2017548376A priority Critical patent/JP2018505911A/ja
Priority to CN201580075472.6A priority patent/CN107405398A/zh
Priority to MX2017007136A priority patent/MX2017007136A/es
Priority to CA2969730A priority patent/CA2969730A1/fr
Priority to EP15865129.9A priority patent/EP3226900A4/fr
Priority to AU2015357463A priority patent/AU2015357463B2/en
Priority to US14/960,973 priority patent/US10370455B2/en
Publication of WO2016090347A1 publication Critical patent/WO2016090347A1/fr
Priority to IL252620A priority patent/IL252620A0/en
Priority to US16/531,968 priority patent/US20200199254A1/en
Priority to JP2021170622A priority patent/JP2022023145A/ja

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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • C07K2319/00—Fusion polypeptide
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the present application generally relates to the identification of the receptor for VISTA (V-region Immunoglobulin-containing Suppressor of T cell Activation f ), a previously identified immunomodulatory polypeptide in the B7 family that regulates T cell activation and proliferation.
  • the invention also relates to the use of the resultant receptor to produce compounds that agonize or antagonize the effects of VSIG8 which may be used as therapeutics, especially in the treatment of cancer, infectious conditions, autoimmunity, inflammation and allergic disorders.
  • the invention specifically relates to the use of the resultant receptor to produce compounds that agonize or antagonize the VISTA/VISTA-R binding interaction.
  • the invention relates to the use of such agonists or antagonists to enhance or inhibit immune functions affected by VISTA such as CD4 + or CD8 + T cell
  • NCR Immune negative checkpoint regulator
  • VISTA V-region Immunoglobulin-containing Suppressor of T cell
  • Activation (1 ) is a recently-identified, NCR ligand, whose closest phyiogenetic relative is PD-L1.
  • VISTA is a ligand that profoundly suppresses immunity (1 )
  • blocking VISTA allows for the development of therapeutic immunity to cancer in pre-clinical oncology models (2).
  • blocking VISTA enhances immunity, especially CD8* and CD4 + mediated T ceil immunity
  • treatment with a soluble Ig fusion protein of the extracellular domain of VISTA suppresses immunity and has been shown to arrest the
  • VISTA is a ligand that induces profound T cell suppression; however, the identity of the receptor that transduces this suppressive effect is currently unknown. Identification of receptors in the field of NCR pathways has been particularly challenging given their extremely low uM affinity and low density.
  • V- R immunoglobulin domain containing 8"as the receptor for VISTA
  • VISTA antagonists e.g., crVISTA mAbs are useful as therapeutics in the treatment of oncology and infection.
  • fragments of VISTA may be used as VISTA antagonists and are potentially useful as therapeutics in the treatment of oncology and infection.
  • VISTA polypeptides e.g., VISTA- lg fusion proteins have been demonstrated to be useful in preventing and treating autoimmunity, inflammation and allergic disorders.
  • VSIG8 will similarly be useful as it will provide a second, independent target for the development of VISTA-R agonists and antagonists, indeed, with regard to receptors in the B7 family, the most effective therapeutics to emerge thus far are (aCTLA4 ⁇ Yervoy ⁇ and aPD-1 ⁇ Novolumab ⁇ ) which both are antibodies that block receptor signaling rather than antibodies to the respective ligands.
  • antibodies to VSIG8 which block or inhibit the VISTAA/ISTA-R interaction should be effective in treating oncology and infectious disease.
  • VISTA-R antagonists will be potentially useful in the treatment of cancer or infectious disease indications such as melanoma and lung cancer or HIV infection.
  • VISTA-R agonists which promote or enhance the VISTA/VISTA-R binding interaction will potentially be useful in the treatment of autoimmune, allergic, and inflammatory indications, GVHD, transplant or other indications wherein the suppression of T cell activation, T cell proliferation or cytokine production is desired.
  • V-R will greatly expedite the clinical development of VISTA-lg as an immunosuppressive drug, and further facilitate the ascertainment of pharmacodynamics, target engagement, and pharmacokinetic studies, which may be used to ascertain the level of receptor occupancy required to produce optimal clinical results.
  • the present invention satisfies these objectives by identifying VSIG8 as the receptor for VISTA.
  • VISTA-R polypeptides VISTA-R fragments and derivatives and VISTA-R fusion proteins that agonize or antagonize the VISTA/VISTA-R binding interaction.
  • VSIG8 agonists or antagonists e.g., agonistic and antagonistic antl-VSIG8 antibodies, and VSIG8 polypeptides, VSIG8 fragments and derivatives and VISTA-R fusion proteins that agonize or antagonize VISTA-R and/or the VISTA/VISTA-R binding interaction to modulate immunity, especially T cell and dendritic cell immunity.
  • VISTA-R agonists or antagonists e.g., agonistic and antagonistic anti-VISTA-R antibodies, and VISTA-R polypeptides, VISTA-R fragments and derivatives and VISTA-R fusion proteins that agonize or antagonize VISTA-R and/or the V!STA/VISTA-R binding interaction to modulate CD4 + or CDS* T cell activation, proliferation, and the production of cytokines.
  • VISTA-R agonists or antagonists e.g., agonistic and antagonistic anti-VISTA-R antibodies, and VISTA-R polypeptides, VISTA-R fragments and derivatives and VISTA-R fusion proteins that agonize or antagonize VISTA-R and/or the VISTA/VISTA-R binding interaction to treat cancer, infectious disease such as viral infection.
  • VISTA-R agonists or antagonists e.g., agonistic and antagonistic anti-VISTA-R antibodies, and VISTA-R polypeptides, VISTA-R fragments and derivatives and VISTA-R fusion proteins that agonize or antagonize VISTA-R and/or the VISTA/VISTA-R binding interaction to treat autoimmune, allergic and inflammatory disease indications.
  • V-R it is a further object of the invention to use the identified V-R to expedite the clinical development of VISTA-lg as an immunosuppressive drug, and facilitate the ascertainment of pharmacodynamics, target engagement, and pharmacokinetic studies and to define the level of receptor occupancy, and dosage required to produce optimal clinical results for VISTA-lg therapeutic compounds.
  • a compound which agonizes or antagonizes the interaction of VISTA and VSIG8 e.g., an antibody or antibody fragment that specifically binds VSIG8, such as an agonistic anti-VSIG8 antibody or antibody fragment or an antagonistic anti-VSIG8 antibody or antibody fragment.
  • a humanized, human, primatized, or chimeric anti-VSIG8 antibody or antibody fragment e.g., a Fab, Fab', scFv or Fabz.
  • a compound which agonizes or antagonizes the interaction of VISTA and VSIG8 e.g., a VSIG8 fusion protein such as a VSIG8 -Ig fusion protein, preferably one comprising a human lgG1 , lgG2, igG3 or lgG4 constant region or fragment thereof, which optionally is mutagenized to eliminate FcR or complement binding or another Fc effector function.
  • VISTA and/or VSIG8 is multimeric, and further wherein the antibody is preferably human, humanized, primatized or chimeric or is a is a Fab, Fab', scFv or Fabs.
  • an antagonist compound according to the invention to inhibit the interaction of VISTA and VSIG8, e.g. to inhibit or block VISTA-associated suppression of T cell activation, proliferation or cytokine production, e.g., to treat a cancer or infectious disease such as a viral, bacterial, protozoan, yeast or fungal, or parasitic disease.
  • It Is another specific object of the invention to provide a diagnostic or therapeutic composition comprising a diagnostica!ly or therapeutically effective amount of an agonist or antagonist compound according to the invention, e.g., which is suitable for use in human therapy, such as an intravenous, subcutaneous or intramuscularly administrable composition.
  • polypeptide or fragment thereof which may be monovalent or multimeric, a PD-1 polypeptide or fragment thereof which may be monovalent or multimeric, or a complex or fusion protein comprising any of the foregoing.
  • an agonist or antagonist compound according to the invention e.g., human immune ceils
  • These assays may be in vitro or use a transgenic animal that expresses human or rodent VISTA and/or human or rodent VSIG8, and may be high throughput screening assays.
  • Figure 1 contains the results of an experiment showing that VISTA inhibits early TCR-induced T cell activation.
  • T ceils were isolated and cultured for 10 minutes with the QCD3 +/- control Ig or VISTA-lg. Total ERK and phosphor-Erk was detected.
  • Figure 2 contains a schematic showing the hierarchy of NCR.
  • Figure 3 contains the results of an experiment showing that VISTA mAb treatment reduces tumor growth.
  • mice were inoculated either sq with A. MB49, B. MCA105, or C. EG7 tumor ceils, or D. i.p with ID8-luciferase tumor cells, and treated with VISTA mAb 13F3 every other day (300 ⁇ tg) beginning on day +1. Subcutaneous tumor growth was monitored. For ID8-iuciferase tumor, mice were imaged on day 30 using Xenogen MS.
  • FIG. 4 contains the results of an experiment showing that therapeutic treatment with mVISTA-lgG2a reverses kidney damage and prolongs survival in the NZBWF1 SLE model.
  • NZBWF1 female mice were treated therapeutically Q3D from 24 weeks of age with PBS (black circles), 150Gg control- lgG2a (blue squares), or 150Og mVISTA-lgG2a (red triangles).
  • (A) Disease severity was monitored weekly by proteinuria. Data is shown as the mean of 6 animals per group ⁇ SEM and is representative of 3 experiments. Statistical significance was determined between control-lgG2a vs. mVISTA-lgG2a; p 0.0156 by the unpaired Mann Whitney test. 5 of 6 VISTA treated mice survived vs zero PBS or lgG2a control treated mice.
  • Figure 5 shows the use of panning to detect PD-1 -+PD-L1 interactions.
  • CHO-S cells were transfected with the indicated GFP-tagged proteins. 48 hours later, cells were cultured on 10ug/ml of plate-bound Fc protein for 1 hour. Nonadherent cells were removed, and bound cells were released with trypsin-EDTA.
  • Figure 6 contains an experiment which shows that VISTA-lg can suppress aCD3 activation of primary human T cells. This data establishes that the
  • upregulation of CD69 in Jurkat T cells can be prevented using VISTA-lg.
  • Figure 7 contains the results of a qPCR assay which compared the expression of VSIG8 in an enriched T cell population relative to a sample containing total spleen cells.
  • Figure 8 contains experiments showing that VISTA fusion proteins specifically bind to VSIG8 over-expressing 293 cells.
  • Figure 9A-C contains experiments showing that antibodies against VSIG8 and VISTA block the interaction between VISTA Ig and VSIG8. .
  • Figure 10 contains experiments indicating that VISTA-lg shows low-level ability to bind to mouse VSIG8 over-expressing cells.
  • Figure 11 contains experiments revealing that VISTA over-expressing cells specifically conjugate to VSIG8 overexpressing cells.
  • Figure 12 contains experiments revealing that antibodies against VSIG8 bind to CDS T cells and NK cells.
  • Figure 13 contains experiments revealing that VSIG8 mRNA is expressed by human CD4 and CDS T cells.
  • Figure 14 contains experiments demonstrating that VISTA signals T cells via VSIG8 to suppress T cell activation.
  • Figure 15 contains experiments showing the upregulation of different immune proteins on VSIG8 expressing A20 cells treated with VISTA-lg including PD- 1.
  • Figure 16 contains a Table identifying different sequences of species orthologs of VSIG8.
  • Activating receptor refers broadly to immune cell
  • T cell receptors that bind antigen, complexed antigen (e.g., in the context of MHC molecules), Ig-fusion proteins, ligands, or antibodies.
  • Activating receptors but are not limited to T cell receptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors, and Fc receptors.
  • T cell receptors are present on T cells and are associated with CDS molecules. T cell receptors are stimulated by antigen in the context of MHC molecules (as well as by polyclonal T cell activating reagents).
  • T cell activation via the ICR results in numerous changes, e.g., protein phosphorylation, membrane lipid changes, ion fluxes, cyclic nucleotide alterations, RNA transcription changes, protein synthesis changes, and cell volume changes.
  • T cell receptors are present on T cells and are associated with CD3 molecules. T cell receptors are stimulated by antigen in the context of MHC molecules (as well as by polyclonal T cell activating reagents).
  • T cell activation via the TCR results in numerous changes, e.g., protein phosphorylation, membrane lipid changes, ion fluxes, cyclic nucleotide alterations, RNA transcription changes, protein synthesis changes, and cell volume changes.
  • Adjuvant refers to an agent used to stimulate the
  • "Aids in the diagnosis” or “aids in the detection” of a disease herein means that the expression level of a particular marker polypeptide or expressed RNA is detected alone or in association with one or more other markers in order to assess whether a subject has cells characteristic of a particular disease condition or the onset of a particular disease condition or comprises immune dysfunction such as immunosuppression characterized by VSIG8 and/or VISTA expression or abnormal immune upregulation characterized by cells having reduced VSIG8 levels, such as during autoimmunity, inflammation or allergic responses, e.g., in individuals with chronic and non-chronic diseases.
  • immune dysfunction such as immunosuppression characterized by VSIG8 and/or VISTA expression or abnormal immune upregulation characterized by cells having reduced VSIG8 levels, such as during autoimmunity, inflammation or allergic responses, e.g., in individuals with chronic and non-chronic diseases.
  • Allergic disease refers broadly to a disease involving allergic reactions. More specifically, an "allergic disease” is defined as a disease for which an allergen is identified, where there is a strong correlation between exposure to that allergen and the onset of pathological change, and where that pathological change has been proven to have an immunological mechanism. Herein, an immunological mechanism means that leukocytes show an immune response to allergen stimulation.
  • Amino acid refers broadly to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mime tics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (e.g., hydroxyproline, y -carboxyglutamate, and 0-phosphoserine. )
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.
  • Analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • T cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal).
  • reexposure of the cells to the same antigen results in failure to produce cytokines and, thus, failure to proliferate.
  • Anergic T cells can, however, mount responses to unrelated antigens and can proliferate if cultured with cytokines (e.g., IL-2).
  • cytokines e.g., IL-2 2
  • T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line.
  • a reporter gene construct can be used.
  • anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL- 2 gene enhancer or by a multimer of the API sequence that can be found within the enhancer (Kang et al. (1992) Science 257: 1134). Modulation of a costimulatory signal results in modulation of effector function of an immune cell.
  • Antibody refers broadly to an "antigen-binding portion” of an antibody (also used interchangeably with “antibody portion,” “antigen- binding fragment,” “antibody fragment”), as well as whole antibody molecules.
  • the term “antibody” as referred to herein includes whole polyclonal and monoclonal antibodies and any antigen-binding fragment (/. e., "antigen-binding portion”) or single chains thereof.
  • an “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen- binding portion thereof.
  • Each heavy chain is comprised of at least one heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, Cm and Cm-
  • Each light chain is comprised of at least one light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL-The VH and VL regions can be further subdivided into regions of hypervariability, termed
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order FR1 , CDR1 , FR2, CDR2, FR3, CDRS, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non- covalent binding interactions stabilize the complex between the molecular structure and the epitope.
  • the archetypal antibody molecule is the
  • immunoglobulin and all types of immunoglobulins, IgG, IgM, igA, IgE, IgD, etc., from ail sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be "antibodies.”
  • the antigen-binding function of an antibody can be performed by
  • Non-limiting examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (a) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (b) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (c) a Fd fragment consisting of the VH and CHI domains; (d) a F v fragment consisting of the VL and VH domains of a single arm of an antibody; (e) a dAb fragment (Ward, et al.
  • VL and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and VH regions pair to form monovalent molecules (known as single chain Fv (scFv).
  • scFv single chain Fv
  • Single chain antibodies are also intended to be encompassed within the term "antigen- binding portion" of an antibody.
  • Any V H and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG molecules or other isotypes.
  • VH and VL can also be used in the generation of Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecifsc antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites. See e.g.
  • an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, et al. (1995) Hum.
  • Antibodies Hybridomas 6: 93-101 and use of a cysteine residue, a marker peptide and a C-terminal polyhlstidine tag to make bivalent and biotinylated scFv molecules. Kipriyanov, et al. (1994) Mol. Immunol. 31 : 1047-1058.
  • Antibody portions, such as Fab and F(ab3 ⁇ 4 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, e.g., humanized, or chimeric antibodies.
  • antigen is used interchangeably herein with the term "an antibody which binds specifically to an antigen” and refers to an immunoglobulin or fragment thereof that specifically binds an antigen.
  • Antigen refers broadly to a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce an antibody capable of binding to an epitope of that antigen.
  • An antigen may have one epitope, or have more than one epitope.
  • the specific reaction referred to herein indicates that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • antigens include, but are not limited to; infectious disease antigens for which a protective immune response may be elicited are exemplary.
  • Antigen presenting ceil refers broadly to professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, and Langerhans cells) as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).
  • professional antigen presenting cells e.g., B lymphocytes, monocytes, dendritic cells, and Langerhans cells
  • other antigen presenting cells e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes.
  • Antisense nucleic acid molecule refers broadly to a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule) complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid molecule can hydrogen bond to a sense nucleic acid molecule.
  • Apoptotic cell death can be characterized by cell shrinkage, membrane blebbing, and chromatin condensation culminating in cell fragmentation. Cells undergoing apoptosis also display a characteristic pattern of internucleosomal DNA cleavage.
  • autoimmune disease or condition refers broadly to a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom, and includes.
  • autoimmune conditions include inflammatory or allergic conditions, e.g., chronic diseases characterized by a host immune reaction against self-antigens potentially associated with tissue destruction such as rheumatoid arthritis.
  • B cell receptor refers broadly to the complex between membrane Ig (mlg) and other transmembrane polypeptides (e.g., IgA. and Ig ) found on B cells.
  • the signal transduction function of mlg is triggered by crosslinking of receptor molecules by oligomeric or multimeric antigens.
  • B cells can also be activated by anti-immunoglobulin antibodies. Upon BCR activation, numerous changes occur in B cells, including tyrosine phosphorylation.
  • Cancer refers broadly to any neoplastic disease
  • cancer or cancerous
  • neoplastic disease whether invasive, non-invasive or
  • metastatic which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor, non-limiting examples of which are described herein. This includes any physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer are exemplified in the working examples. Further cancers include but are not limited to, carcinoma, lymphoma, b!astoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non- cle
  • cancers amenable for treatment by the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include colorectal, bladder, ovarian, melanoma, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • PTLD post-transplant lymphoproliferative disorder
  • the cancer is selected from the group consisting of colorectal cancer, breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma.
  • the cancer is an early or advanced (including metastatic) bladder, ovarian or melanoma.
  • the cancer is colorectal cancer.
  • the cancerous conditions amenable for treatment of the invention include cancers that express or do not express VSIG8 and/or VISTA and further include non-metastatic or non-invasive as well as invasive or metastatic cancers wherein VSIG8 and/or VISTA expression by immune, stroma or diseased cells suppress antitumor responses and anti-invasive immune responses.
  • the method of the present invention is particularly suitable for the treatment of vascularized tumors.
  • Cancers according to the invention include cancers that express or do not express VSIG8 and/or VISTA and further include non-metastatic or non-invasive as well as invasive or metastatic cancers wherein VSIG8 and/or VISTA expression by immune, stromal or diseased cells suppress antitumor responses and anti-invasive immune responses, and those characterized by vascularized tumors.
  • Cancer therapy herein refers to any method which prevents or treats cancer or ameliorates one or more of the symptoms of cancer.
  • Such therapies will comprise the administration of an immunostimulatory VSIG8 and/or VISTA polypeptide or fusion protein, conjugate, muitimer (homomultimer or heteromuitimer) or composition containing according to the invention either alone or more typically in combination with chemotherapy or radiotherapy or other biologies and for enhancing the activity thereof, /. e., in individuals wherein VSIG8 and/or VISTA expression suppress antitumor responses and the efficacy of chemotherapy or radiotherapy or biologic efficacy.
  • Any chemotherapeutic agent exhibiting anticancer activity can be used according to the present invention.
  • the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids,
  • epipodophy!lotoxins antibiotics, L- Asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroids, progestins, estrogens, antiestrogen, androgens, antiandrogen, and
  • the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxal ' iplatin, capecitabine, paclitaxel and docetaxel.
  • 5-fluorouracil 5-FU
  • LV leucovorin
  • irinotecan oxal ' iplatin
  • capecitabine paclitaxel
  • paclitaxel paclitaxel
  • docetaxel Two or more chemotherapeutic agents can be used in a cocktail to be administered in combination with administration of the anti-VEGF antibody.
  • One preferred combination chemotherapy is fluorouracil-based, comprising 5-FU and one or more other chemotherapeutic agent(s). Suitable dosing regimens of combination chemotherapies are known in the art and described in, for example, Saltz et al.
  • the biologic may be another immune potentiator such as antibodies to PD-L1 , PD- L2, CTLA-4, or VISTA as well as PD-L1 , PD-L2, CTLA-4 or VISTA fusion proteins as well as cytokines, growth factor antagonists and agonists, hormones and anti-cytokine antibodies.
  • the constant region, or a portion thereof is altered, replaced or exchanged so that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, the variable region or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • an enzyme, toxin, hormone, growth factor, drug, the variable region or a portion thereof is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • Codon region refers broadly to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term “noncoding region” refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).
  • Constantly modified variants applies to both amino acid and nucleic acid sequences, and with respect to particular nucleic acid sequences, refers broadly to conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. "Silent variations” are one species of conservatively modified nucleic acid variations. Every nucleic acid sequence herein which encodes a
  • polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) may be modified to yield a functionally identical molecule.
  • Complementarity determining region refers broadly to one or more of the hyper-variable or
  • CDRs complementarily determining regions found in the variable regions of light or heavy chains of an antibody.
  • CDRs complementarily determining regions
  • the CDRs in each chain are held in close proximity by framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site.
  • selectivity determining regions SDRs
  • Control amount refers broadly to a marker can be any amount or a range of amounts to be compared against a test amount of a marker.
  • a control amount of a marker may be the amount of a marker in a patient with a particular disease or condition or a person without such a disease or condition.
  • a control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
  • Costimuiatory receptor refers broadly to receptors
  • inhibitory receptors includes receptors which transmit a negative signal to an immune cell, e.g., a T cell or an NK cell.
  • Couple refers broadly to the ability of a
  • costimuiatory molecule to provide a second, non-activating, receptor-mediated signal (a "costimuiatory signal") that induces proliferation or effector function.
  • a costimuiatory signal can result in cytokine secretion (e.g., in a T cell that has received a T cell-receptor-mediated signal)
  • Immune cells that have received a cell receptor-mediated signal may be referred to herein as “activated immune cells.” With respect to T cells,
  • a costimuiatory signal can induce cytokine secretion (e.g., IL-2 and/or IL-10) in a T cell and/or can prevent the induction of unresponsiveness to antigen, the induction of anergy, or the induction of cell death in the T cell.
  • cytokine secretion e.g., IL-2 and/or IL-10
  • Costimuiatory polypeptide or “costimuiatory molecule” herein refers to a polypeptide that, upon interaction with a cell-surface molecule on T cells, modulates T cell responses.
  • Costimuiatory signaling is the signaling activity resulting from the interaction between costimuiatory polypeptides on antigen presenting cells and their receptors on T ceils during antigen-specific T cell responses. Without wishing to be limited by a single hypothesis, the antigen-specific T cell response is believed to be mediated by two signals: 1 ) engagement of the T cell Receptor (TCR) with antigenic peptide presented in the context of MHC (signal 1 ), and 2) a second antigen-independent signal delivered by contact between different costimulatory receptor/ligand pairs (signal 2).
  • TCR T cell Receptor
  • signal 2 a second antigen-independent signal delivered by contact between different costimulatory receptor/ligand pairs
  • this "second signal” is critical in determining the type of T cell response (activation vs inhibition) as well as the strength and duration of that response, and is regulated by both positive and negative signals from costimulatory molecules, such as the B7 family of proteins.
  • polypeptide herein means a member of the B7 family of proteins that costimulate T cells including, but not limited to B7-1 , B7-2, B7-DC, B7-H5, B7-HI, B7-H2, B7-H3, B7-H4, B7-H6, B7-S3 and biologically active fragments and/or variants thereof.
  • Representative biologically active fragments include the extracellular domain or fragments of the extracellular domain that costimulate T ceils.
  • Cytoplasmic domain refers broadly to the portion of a protein which extends into the cytoplasm of a cell.
  • Diagnostic refers broadly to identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay are termed “true negatives.”
  • the "specificity” of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • Diagnosing or “aiding in the diagnosis” as used herein refers broadly to classifying a disease or a symptom, and/or determining the likelihood that an individual has a disease condition (e.g., based on absence or presence of VSIG8 and/or VISTA expression, and/or increased or decreased expression by immune, stromal and/or putative diseased cells); determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • the term "detecting” may also optionally encompass any of the foregoing.
  • Diagnosis of a disease according to the present invention may, in some embodiments, be affected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease.
  • a biological sample obtained from the subject may also optionally comprise a sample that has not been physically removed from the subject.
  • Effective amount refers broadly to the amount of a
  • the effective amount may be an amount effective for prophylaxis, and/or an amount effective for prevention.
  • the effective amount may be an amount effective to reduce, an amount effective to prevent the incidence of signs/symptoms, to reduce the severity of the incidence of signs/symptoms, to eliminate the incidence of signs/symptoms, to slow the development of the incidence of signs/symptoms, to prevent the development of the incidence of
  • the “effective amount” may vary depending on the disease and its severity and the age, weight, medical history, susceptibility, and pre-existing conditions, of the patient to be treated.
  • the term “effective amount” is synonymous with “therapeutically effective amount” for purposes of this invention.
  • “Expression vector,” as used herein, refers broadly to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell.
  • the term includes linear or circular expression systems.
  • the term includes expression systems that remain episomal or integrate into the host cell genome.
  • the expression systems can have the ability to self -replicate or not, I. e., drive only transient expression in a cell.
  • the term includes recombinant expression cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.
  • “Family,” as used herein, refers broadly to the polypeptide and nucleic acid molecules of the invention is intended to mean two or more polypeptide or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • Family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a family can contain a first polypeptide of human origin, as well as other, distinct polypeptides of human origin or alternatively, can contain homologues of non-human origin (e.g., monkey polypeptides.)
  • Members of a family may also have common functional characteristics.
  • Tc receptor * refers broadly to cell surface
  • Fc receptors for the Fc portion of immunoglobulin molecules are found on many cells which participate in immune responses. Among the human FcRs that have been identified so far are those which recognize IgG (designated FcyR), IgE (FceRI), IgA (FcaR), and polymerized IgM/A (FcepR). FcRs are found in the following cell types: FceRI (mast cells), FceRI I (many leukocytes), FcaR (neutrophils), and FcpR (glandular epithelium, hepatocytes). (Hogg Immunol. Today 9: 185-86 (1988)).
  • the widely studied FcyRs are central in cellular immune defenses, and are responsible for stimulating the release of mediators of inflammation and hydrolytic enzymes involved in the pathogenesis of autoimmune disease. (Unkeless, Annu. Rev. Immunol. 6: 251-87 (1988)).
  • the FcyRs provide a crucial link between effector cells and the lymphocytes that secrete Ig, since the macrophage/monocyte, polymorphonuclear leukocyte, and natural killer (NK) cell FcyRs confer an element of specific recognition mediated by IgG.
  • Human leukocytes have at least three different receptors for IgG: hFcpRI (found on monocytes/macrophages), hFcyRII (on monocytes, neutrophils, eosinophils, platelets, possibly B ceils, and the K562 cell line), and Fcyili (on NK cells, neutrophils, eosinophils, and macrophages).
  • hFcpRI found on monocytes/macrophages
  • hFcyRII on monocytes, neutrophils, eosinophils, platelets, possibly B ceils, and the K562 cell line
  • Fcyili on NK cells, neutrophils, eosinophils, and macrophages.
  • Framework region or "FR,” as used herein refers broadly to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody. See Kabat, et al. "Sequences of Proteins of
  • These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.
  • Heterologous refers broadly to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid (e.g., a promoter from one source and a coding region from another source.)
  • a heterologous protein indicates that the protein comprises two or more
  • subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • High affinity refers broadly to an antibody or fusion
  • "high affinity" binding can vary for different antibody isotypes.
  • "high affinity" binding for an IgM isotype refers to an antibody having a KD of 10 -7 M or less, more preferably 10 -8 M or less.
  • Homology may be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is one that is not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be
  • Host cell refers broadly to refer to a cell into which a nucleic acid molecule of the invention, such as a recombinant expression vector of the invention, has been introduced.
  • Host cells may be prokaryotic cells (e.g., E. cols), or eukaryotic cells such as yeast, insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa, HEK-293, e.g., cultured cells, explants, and cells in vivo.
  • host cell and "recombinant host ceil” are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • Human monoclonal antibody refers to antibodies displaying a single
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • a transgenic nonhuman animal e.g., a transgenic mouse
  • Humanized antibody refers broadly to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences.
  • the humanized antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
  • the term "humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Hybridization refers broadly to the physical interaction of complementary (including partially complementary) polynucleotide strands by the formation of hydrogen bonds between complementary nucleotides when the strands are arranged antiparallel to each other.
  • IgV domain and IgC domain refer broadly to Ig
  • Ig folds are comprised of a sandwich of two ⁇ sheets, each consisting of antiparallel ⁇ strands of 5-10 amino acids with a conserved disulfide bond between the two sheets in most, but not all, domains.
  • IgC domains of ig, TCR, and MHC molecules share the same types of sequence patterns and are called the CI set within the Ig superfamily. Other IgC domains fall within other sets.
  • IgV domains also share sequence patterns and are called V set domains. IgV domains are longer than C-domains and form an additional pair of ⁇ strands.
  • Immunune cell refers broadly to cells that are of
  • Immune cells include but are not limited to lymphocytes, such as 8 cells and T cells; natural killer cells; dendritic cells, and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • Immunoassay refers broadly to an assay that uses an antibody to specifically bind an antigen.
  • the immunoassay may be characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • Immunorelated disease or disorder or condition
  • Immuno response refers broadly to T cell-mediated and/or B cell-mediated immune responses that are influenced by modulation of T cell costimulation.
  • Exemplary immune responses include 8 cell responses (e.g., antibody production) T cell responses (e.g., cytokine production, and cellular cytotoxicity) and activation of cytokine responsive cells, e.g.,
  • the term “downmodulation” with reference to the immune response includes a diminution in any one or more immune responses, while the term “upmodulation” with reference to the immune response includes an increase in any one or more immune responses. It will be understood that upmodulation of one type of immune response may lead to a corresponding downmodulation in another type of immune response. For example,
  • cytokines e.g., IL-10
  • upmodulation of the production of certain cytokines can lead to downmodulation of cellular immune responses.
  • Immunologic refers to the development of a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient.
  • a humoral antibody mediated
  • a cellular response mediated by antigen-specific T cells or their secretion products
  • Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells.
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class II or Class I MHC molecules to activate antigen-specrfic CD4* T helper cells and/or CD8 + cytotoxic T cells, respectively.
  • the response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity.
  • the presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 + T cells) or CTL (cytotoxic T lymphocyte) assays.
  • proliferation assays CD4 + T cells
  • CTL cytotoxic T lymphocyte
  • Infectious agent herein refers to any pathogen or agent that infects
  • mammalian cells preferably human cells and causes a disease condition.
  • infectious agents include by way of example those involved in (a) viral diseases such as, for example, diseases resulting from infection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e ⁇ g-, an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picornavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenza virus), a paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a cononavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those
  • hepadnavirus e.g., hepatitis B virus
  • flavi virus e.g., hepatitis C virus or Dengue virus
  • retrovirus e.g., a lentivirus such as HIV
  • bacterial diseases such as, for example, diseases resulting from infection by bacteria of, for example, the genus Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella; (c) other infectious diseases, such as bacterial diseases
  • infectious agent antigen herein means a compound, e.g., peptide
  • the antigen will comprise a moiety, e.g., polypeptide or glycoprotein expressed on the surface of the virus or other infectious agent, such as a capsid protein or other membrane protein.
  • Inflammatory bowel disease herein comprises any inflammatory bowel condition and especially includes inflammatory bowel disease, Crohn's disease, ulcerative colitis (UC), collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's disease, and indeterminate colitis.
  • inflammatory bowel disease Crohn's disease, ulcerative colitis (UC), collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's disease, and indeterminate colitis.
  • Inflammatory disorders refers broadly to chronic or acute inflammatory diseases, and expressly includes inflammatory autoimmune diseases and inflammatory allergic conditions. These conditions include by way of example inflammatory abnormalities characterized by dysregulated immune response to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammatory disorders underlie a vast variety of human diseases. Non-immune diseases with etiological origins in inflammatory processes include cancer, atherosclerosis, and ischemic heart disease.
  • disorders associated with inflammation include: Chronic prostatitis, Glomerulonephritis, Hypersensitivities, Pelvic inflammatory disease, Reperfusion injury, Sarcoidosis, Vasculitis, interstitial cystitis, normocompiementemic urticarial vasculitis, pericarditis, myositis, anti- synthetase syndrome, scleritis, macrophage activation syndrome, Behcet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult and juvenile Still's disease, cryropyrinopathy, Muckle- Wells syndrome, familial cold-induced auto- inflammatory syndrome, neonatal onset multisystemic inflammatory disease, familial Mediterranean fever, chronic infantile neurologic, cutaneous and articular syndrome, systemic juvenile idiopathic arthritis, Hyper IgD syndrome,
  • TRAPS P TNF receptor-associated periodic syndrome
  • Periodontitis periodontitis
  • hepatitis cirrhosis
  • pancreatitis myocarditis
  • vasculitis gastritis
  • gastritis gout
  • gouty arthritis and inflammatory skin disorders, selected from the group consisting of psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.
  • Inhibitory signal refers broadly to a signal transmitted via an inhibitory receptor molecule on an immune cell.
  • a signal antagonizes a signal via an activating receptor (e.g., via a TCR, CDS, BCR, or Fc molecule) and can result, e.g., in inhibition of: second messenger generation; proliferation; or effector function in the immune cell, e.g., reduced phagocytosis, antibody production, or cellular cytotoxicity, or the failure of the immune ceil to produce mediators (e.g., cytokines (e.g., IL-2) and/or mediators of allergic responses); or the development of anergy.
  • an activating receptor e.g., via a TCR, CDS, BCR, or Fc molecule
  • Isolated material may be, for example, exogenous nucleic acid included in a vector system, exogenous nucleic acid contained within a host cell, or any material which has been removed from its original environment and thus altered by the hand of man (e.g., "isolated antibody”).
  • isolated antibody any material which has been removed from its original environment and thus altered by the hand of man.
  • purified refers broadly to a protein, DNA, antibody, RNA, or biologically active portion thereof, that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the biological substance is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • purified refers broadly to a protein, DNA, antibody, RNA, or biologically active portion thereof, that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the biological substance is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • isolated refers to a compound of interest (for example a polynucleotide or a polypeptide) that is in an environment different from that in which the compound naturally occurs e.g., separated from its natural milieu such as by concentrating a peptide to a concentration at which it is not found in nature.
  • isolated includes compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.
  • isolated antibody is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds VSIG8 and/or VISTA) is substantially free of antibodies that specifically bind antigens other than VSIG8 ). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • isotype herein refers to the antibody class (e.g., IgM or !gG1 ) that is encoded by the heavy chain constant region genes.
  • K-assoc or "K a ". as used herein, refers broadly to the association rate of a particular antibody-antigen interaction, whereas the term “Kdiss” or “Kd,” as used herein, refers to the dissociation rate of a particular antibody-antigen interaction.
  • KQ is intended to refer to the dissociation
  • K D values for antibodies can be determined using methods well established in the art such as piasmon resonance (BIAcore®), ELISA and KINEXA.
  • a preferred method for determining the KD of an antibody is by using surface Piasmon resonance, preferably using a biosensor system such as a BIAcore® system or by ELISA.
  • Label or a “detectable moiety” as used herein, refers broadly to a
  • composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • Low stringency refers broadly to conditions for nucleic acid hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Ausubel, et ai., Short Protocols in Molecular Biology (5 th Ed.) John Wiley & Sons, NY (2002).
  • Exemplary specific hybridization conditions include but are not limited to: (1 ) low stringency hybridization conditions in 6 X sodium chloride/sodium citrate (SSC) at about 45° C, followed by two washes in 0. 2XSSC, 0.1 % SDS at least at 50°C. (the temperature of the washes can be increased to 55 °C.
  • low stringency conditions (2) medium stringency hybridization conditions in 6XSSC at about 45°C, , followed by one or more washes in 0. 2X SSC, 0. 1% SDS at 60°C. ; (3) high stringency hybridization conditions in 6XSSC at about 45°C followed by one or more washes in 0. 2X. SSC, 0. 1 % SDS at 65°C; and (4) very high stringency hybridization conditions are 0. 5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2XSSC, and 1 % SDS at 65° C.
  • mammal refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, miik-producing mammary glands for nourishing the young.
  • mammals include but are not limited to alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non- human primates, pigs, rats, sheep, shrews, squirrels, tapirs, and voles.
  • Mammals include but are not limited to bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington D. C.
  • Naturally-occurring nucleic acid molecule refers broadly refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • Nucleic acid or “nucleic acid sequence,” as used herein, refers broadly to a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form.
  • the term encompasses nucleic acids, i.e.,
  • oligonucleotides containing known analogs of natural nucleotides.
  • the term also encompasses nucleic- acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence also implicitly
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • Oligomerization domain or “mu!timerization domain” or “dimerization domain” are used interchangeably herein, and refer broadly to a domain that when attached to a VSIG8 and/or VISTA extracellular domain or fragment thereof, facilitates oligomerization.
  • Said oligomerization domains comprise self- associating, a -helices, for example, leucine zippers, that can be further stabilized by additional disulfide bonds.
  • the domains are designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein.
  • coiled GCN4 examples thereof are known in the art and include by way of example coiled GCN4, and COMP.
  • the a-helical coiled coil is probably the most widespread subunit oiigomerization motif found in proteins. Accordingly, coiled coils fulfill a variety of different functions. In several families of transcriptional activators, for example, short leucine zippers play an important role in positioning the DNA- binding regions on the DNA. Ellenberger, et ai. (1992) Ce/// 71 : 1223-1237.
  • Coiled coils are also used to form oligomers of intermediate filament proteins. Coiled-coil proteins furthermore appear to play an important role in both vesicle and viral membrane fusion. (Skehel and Wiley Cell 95: 871-874(1998)). In both cases hydrophobic sequences, embedded in the membranes to be fused, are located at the same end of the rod-shaped complex composed of a bundle of long a-helices. This molecular arrangement is believed to cause close
  • the coiled coil is often used to control oiigomerization. It is found in many types of proteins, including transcription factors include, but not limited to GCN4, viral fusion peptides, SNARE complexes and certain tRNA synthetases, among others. Very long coiled coils are found in proteins such as tropomyosin, intermediate filaments and spindle -pole-body components. Coiled coils involve a number of a-helices that are supercoiled around each other in a highly organized manner that associate in a parallel or an antiparallel orientation.
  • the helices may be from the same or from different proteins.
  • the coiled-coil is formed by component helices coming together to bury their hydrophobic seams. As the hydrophobic seams twist around each helix, so the helices also twist to coil around each other, burying the hydrophobic seams and forming a supercoil. It is the characteristic interdigitation of side chains between neighboring helices, known as knobs-into- holes packing, that defines the structure as a coiled coil.
  • the helices do not have to run in the same direction for this type of interaction to occur, although parallel conformation is more common.
  • Antiparallel conformation is very rare in trimers and unknown in pentamers, but more common in intramolecular dimers, where the two helices are often connected by a short loop.
  • the heterotrimeric coiled-coil protein laminin plays an important role in the formation of basement membranes.
  • Other examples are the thrombospondins and cartilage oligomeric matrix protein (COMP) in which three (thrombospondins 1 and 2) or five (thrombospondins 3, 4 and COMP) chains are connected.
  • the molecules have a flower bouquet-like appearance, and the reason for their oligomeric structure is probably the multivalent interaction of the C-terminal domains with cellular receptors.
  • the yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. Ellenberger, et al. Cell 71 : 1223-1237 (1992).
  • the bZIP dimer is a pair of continuous a helices that form a parallel coiled-coil over their carboxy-terminal 34 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA binding site.
  • the coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T.
  • bZIP contains a 4-3 heptad repeat of hydrophobic and nonpolar residues that pack together in a parallel a-helical coiled-coil.
  • the stability of the dimer results from the side-by-side packing of leucines and nonpolar residues in positions a and d of the heptad repeat, as well as a limited number of intra- and interhelical salt bridges, shown in a crystal structure of the GCN4 leucine zipper peptide.
  • CMP ce// 71 : 1223-1237 (1992)
  • Another example is CMP (matrilin-1 ) isolated from bovine tracheal cartilage as a homotrimer of subunits of Mr 52,000 (Pau!sson & Heinegard (1981 ), Biochem. J. 197: 367- 375), where each subunit consists of a vWFAI module, a single EGF domain, a vWFA2 module and a coiled coil domain spanning five heptads.
  • vWFAI single EGF domain
  • vWFA2 vWFA2
  • COMP non-co!lagenous glycoprotein
  • the protein is a 524 kDa homopentamer of five subunits which consists of an N-terminal heptad repeat region (cc) followed by four epidermal growth factor (EGF)-like domains (EF), seven calcium-binding domains (T3) and a C-terminal globular domain (TC).
  • EGF epidermal growth factor
  • T3 calcium-binding domains
  • TC C-terminal globular domain
  • COMP belongs to the family of thrombospondins.
  • fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • Paratope refers broadly to the part of an antibody which recognizes an antigen (e.g., the antigen-binding site of an antibody.) Paratopes may be a small region (e.g., 15-22 amino acids) of the antibody's Fv region and may contain parts of the antibody's heavy and light chains. See Goldsby, et al. Antigens (Chapter 3) Immunology (5 th Ed.) New York: W. H. Freeman and Company, pages 57-75.
  • Patient or “subject” or “recipient”, “individual”, or “treated individual” are used interchangeably herein, and refers broadly to any animal that is in need of treatment either to alleviate a disease state or to prevent the occurrence or reoccurrence of a disease state.
  • “Patient” as used herein refers broadly to any animal that has risk factors, a history of disease, susceptibility, symptoms, and signs, was previously diagnosed, is at risk for, or is a member of a patient population for a disease.
  • the patient may be a clinical patient such as a human or a veterinary patient such as a companion, domesticated, livestock, exotic, or zoo animal.
  • Polypeptide “peptide” and “protein,” are used interchangeably and refer broadly to a polymer of amino acid residues s of any length, regardless of modification (e.g., phosphorylation or glycosylation).
  • the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a
  • Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
  • the terms "polypeptide,” “peptide” and “protein” expressly include glycoproteins, as well as non-glycoproteins.
  • Promoter refers broadly to an array of nucleic acid
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a constitutive" promoter is a promoter that is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
  • prophylactically effective amount refers broadly to the amount of a compound that, when administered to a patient for prophylaxis of a disease or prevention of the reoccurrence of a disease, is sufficient to effect such prophylaxis for the disease or reoccurrence.
  • the prophylactically effective amount may be an amount effective to prevent the incidence of signs and/or symptoms.
  • the “prophylactically effective amount” may vary depending on the disease and its severity and the age, weight, medical history, predisposition to conditions, preexisting conditions, of the patient to be treated.
  • prophylactic vaccine and/or “Prophylactic vaccination” refers to a vaccine used to prevent a disease or symptoms associated with a disease such as cancer or an infectious condition.
  • Prophylaxis refers broadly to a course of therapy where signs and/or symptoms are not present in the patient, are in remission, or were previously present in a patient. Prophylaxis includes preventing disease occurring subsequent to treatment of a disease in a patient. Further, prevention includes treating patients who may potentially develop the disease, especially patients who are susceptible to the disease (e.g., members of a patent population, those with risk factors, or at risk for developing the disease).
  • Recombinant refers broadiy with reference to a product, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the ceil is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant,
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germ!ine immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animai transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • Signal sequence refers broadly to a peptide containing about 15 or more amino acids which occurs at the N-terminus of secretory and membrane bound polypeptides and which contains a large number of hydrophobic amino acid residues.
  • a signal sequence contains at least about 10-30 amino acid residues, preferably about 15-25 amino acid residues, more preferably about 18-20 amino acid residues, and even more preferably about 19 amino acid residues, and has at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g., Valine, Leucine, Isoleucine or Phenylalanine).
  • a “signal sequence,” also referred to in the art as a “signal peptide,” serves to direct a polypeptide containing such a sequence to a lipid bilayer, and is cleaved in secreted.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than about 10 to 100 times background.
  • nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art. (See, e.g., Turner, et al. CSH Symp. Quant Biol. Lit 123-33 (1987); Frier, et al. PNAS 83: 9373-77 1986); Turner, et al. J. Am. Chem.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., about at least 5, 6, 7, 8, 9, 10 out of 10 being about at least 50%, 60%, 70%, 80%, 90%, and 100% complementary, inclusive).
  • Perfectly complementary or 100% complementarity refers broadly all of the contiguous residues of a nucleic acid sequence hydrogen bonding with the same number of contiguous residues in a second nucleic acid sequence.
  • Substantial complementarity refers to polynucleotide strands exhibiting about at least 90% complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected so as to be noncomplementary. Specific binding requires a sufficient degree of complementarity to avoid nonspecific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, /. under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.
  • the non- target sequences typically may differ by at least 5 nucleotides.
  • Solid support refers broadly to any materia! that provides a solid or semi-solid structure with which another material can be attached including but not limited to smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) as well as textured and porous materials.
  • smooth supports e.g., metal, glass, plastic, silicon, and ceramic surfaces
  • Soluble ectodomain (ECD)" or “ectodomain” or "soluble VSIG8 or VISTA protein(s)/molecule(s)" of VSIG8 and/or VISTA as used herein means non-cell- surface-bound VSIG8 and/or VISTA molecules or any portion thereof, including, but not limited to: VSIG8 and/or VISTA fusion proteins or VSIG8 and/or VISTA ECD-lg fusion proteins, wherein the extracellular domain of VSIG8 and/or VISTA or fragment thereof is fused to an immunoglobulin (Ig) moiety rendering the fusion molecule soluble, or fragments and derivatives thereof, proteins with the extracellular domain of VSIG8 and/or VISTA fused or joined with a portion of a biologically active or chemically active protein such as the papillomavirus E7 gene product, melanoma-associated antigen p97 or HIV env protein, or fragments and derivatives thereof; hybrid
  • soluble VSIG8 or VISTA protein(s)/molecule ⁇ s) also include VSIG8 or VISTA molecules with the transmembrane domain removed to render the protein soluble, or fragments and derivatives thereof; fragments, portions or derivatives thereof, and soluble VSIG8 or VISTA mutant molecules.
  • the soluble VSIG8 or VISTA molecules used in the methods according to at least some embodiments of the invention may or may not include a signal (leader) peptide sequence.
  • Subject or “patient” or “individual” in the context of therapy or diagnosis herein includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc., i.e., anyone suitable to be treated according to the present invention include, but are not limited to, avian and mammalian subjects, and are preferably
  • mammalian Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects of both genders and at any stage of development (i. e., neonate, infant, juvenile, adolescent, and adult) can be treated according to the present invention.
  • the present invention may also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, cattle, goats, sheep, and horses for veterinary purposes, and for drug screening and drug development purposes. "Subjects" is used
  • substantially free of chemical precursors or other chemicals refers broadly to preparations of VSIG8 or VISTA protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes
  • VSIG8 or VISTA protein having less than about 30% (by dry weight) of chemical precursors or non-VSIG8 chemicals, more preferably less than about 20% chemical precursors or non-VSIG8 or VISTA chemicals, still more preferably less than about 10% chemical precursors or non- VSIG8 or VISTA chemicals, and most preferably less than about 5% chemical precursors or non-VSIG8 or VISTA chemicals.
  • "Symptoms" of disease as used herein, refers broadly to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease.
  • T cell refers broadly to CD4 * T cells and CDS* T cells.
  • T cell also includes both T helper 1 type T cells and T helper 2 type T cells.
  • Treatment refers broadly to treating a disease, arresting, or reducing the development of the disease or its clinical symptoms, and/or relieving the disease, causing
  • Therapy encompasses prophylaxis, treatment, remedy, reduction, alleviation, and/or providing relief from a disease, signs, and/or symptoms of a disease. Therapy encompasses an alleviation of signs and/or symptoms in patients with ongoing disease signs and/or symptoms (e.g., inflammation, pain). Therapy also encompasses
  • Treatment refers broadly to the clinical significant reduction in signs and/or symptoms.
  • Therapy includes treating relapses or recurrent signs and/or symptoms (e.g., inflammation, pain). Therapy encompasses but is not limited to precluding the appearance of signs and/or symptoms anytime as well as reducing existing signs and/or symptoms and eliminating existing signs and/or symptoms.
  • Therapy includes treating chronic disease (“maintenance") and acute disease.
  • treatment includes treating or preventing relapses or the recurrence of signs and/or symptoms (e.g., inflammation, pain).
  • Therapeutic vaccine and/or “therapeutic vaccination” refers to a vaccine used to treat a disease such as cancer or an infectious condition.
  • Treg cell (sometimes also referred to as suppressor T cells or inducible Treg cells or iTregs) as used herein refers to a subpopulation of T cells which modulate the immune system and maintain tolerance to self-antigens and can abrogate autoimmune diseases. Foxp3 + CD4 + CD25 + regulatory T cells (Tregs) are critical in maintaining peripheral tolerance under normal
  • Transmembrane domain refers broadly to an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an a-helical structure. In an embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zakla, et al. Annu. Rev. Neurosci. 19:235-263 (1996).
  • Transgenic animal refers broadly to a non-human
  • transgenic animal preferably a mammal, more preferably a mouse, in which one or more of the cells of the animal includes a "transgene".
  • transgene refers to exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, for example directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • Tumor refers broadly to at least one cell or cell mass in the form of a tissue neoformation, in particular in the form of a spontaneous, autonomous and irreversible excess growth, which is more or less disinhibited, of endogenous tissue, which growth is as a rule associated with the more or less pronounced loss of specific cell and tissue functions.
  • This cell or cell mass is not effectively inhibited, in regard to its growth, by itself or by the regulatory mechanisms of the host organism, e.g., colorectal cancer, melanoma or carcinoma.
  • Tumor antigens not only include antigens present in or on the malignant cells themselves, but also include antigens present on the stromal supporting tissue of tumors including endothelial cells and other blood vessel components.
  • immune cells to stimulation, e.g., and stimulation via an activating receptor or a cytokine.
  • Unresponsiveness can occur, e.g., because of exposure to
  • Vaccine refers to a biological preparation that as
  • a vaccine typically includes an adjuvant as immune potentiator to stimulate the immune system. This includes prophylactic ⁇ which prevent disease) and therapeutic vaccines (which treat the disease or its symptoms).
  • Variable region refers broadly to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • Each light chain has a variable domain (V L ) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain,
  • Vector refers broadly to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector is another type of vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacteria! vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Vectors are referred to herein as "recombinant expression vectors” or simply "expression vectors", in general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses,
  • adenoviruses and adeno-associated viruses which serve equivalent functions.
  • the techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook, et al. Molec. Cloning: Lab. Manual [3rd Ed] Cold Spring Harbor Laboratory Press (2001 ). Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture, and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the ig superfamily comprises many critical immune regulators, including the 87 family ligands and receptors. Of these, the best characterized co-stimulatory ligands are CD80 and CD86 who are expressed on professional antigen-presenting cells (APCs) and whose receptors are CD28, PD-L1 and CTLA-4 (12). These targets have proven to be valuable clinical targets in the treatment of autoimmunity and cancer.
  • APCs professional antigen-presenting cells
  • the B7 family ligands have expanded to include co-stimulatory B7-H2 (ICOS Ligand) and B7-H3 (receptor unknown), as well as co-inhibitory B7-H1 (PD- L1 ), B7-DC (PD-L2), B7-H4 (B7S1 or B7x; receptor unknown), B7-H5 (only human)13 and B7-H6 (12, 14).
  • B7-L1 -»PD-1 interactions, CTLA-4 and it engagement by its ligands, B7-1 and B7-2 have all empowered the pharmaceutical and biotechnology industries to predict outcomes, strategically design PK and PD studies, and understand toxicities associated with therapeutic intervention.
  • VISTA is a negative checkpoint regulator (NCR) ligand, whose nearest phylogenetic relative is PD-L1.
  • NCR negative checkpoint regulator
  • VISTA has been shown to profoundly suppress the activation of resting T cells. Both in the human and mouse, VISTA expression appears to be hematopoietica!!y restricted and abundantly expressed in the myeloid compartment, and to a lower intensity on T ceils (16).
  • Blocking VISTA like blocking PD-L1 and PD-1 , enhances immunity in murine models of cancer (17).
  • Antibodies that block or inhibit the effects of VISTA may be used to enhance human immune responses, in particular immune responses to malignancies and infection.
  • molecules that agonize VISTA such as soluble VISTA, e.g., VISTA-lg, may be used to suppress undesired human immune responses such as autoimmune, allergic or inflammatory immune responses.
  • the receptor for VISTA Prior to the present invention, the receptor for VISTA has not been reported. It is anticipated that the identification of this receptor as herein disclosed will facilitate the design of other therapeutics for treating oncology, infectious disease, autoimmune, allergic and inflammatory indications. As well the identification of the receptor will facilitate a greater understanding of the manner that VISTA in association with its receptor modulates immunity.
  • the objective of the present invention was to identify the receptor for VISTA.
  • compounds which antagonize this receptor or which modulate (inhibit or block) the binding of this receptor to VISTA should be useful in treating conditions wherein upregulation of T or NK cell immunity is desirable such as cancer or infectious disease conditions.
  • compounds which agonize this receptor or which modulate (promote or enhance) the binding of this receptor to VISTA should be useful in treating conditions wherein downregu!ation of T or NK cell immunity is desirable such as autoimmunity, allergy, inflammation, GVHD, transplant or cell or gene therapy.
  • the VISTA receptor was predicted by the inventors to be a member of the B7 family as all identified members of the B7 receptor family are members of the Ig superfamily.
  • This Ig superfamily includes proteins that are both soluble and membrane proteins that are involved in cell-cell signaling/interaction, adhesion and communication. All of these molecules contain domains that are similar to those domains of Ig V regions (IgV) or constant regions (IgC).
  • the domains are typically 70-110 amino acids in length, have characteristic Ig folds, which has a sandwich-like structure formed by two a parallel sheets which are formed between cysteine residues in the B and F strands and stabilize the Ig-fold.
  • B7 receptors (CTLA-4, ICOS, PD-1 ) have one IgV domain, in contrast to the ligands for these receptors (CD80, CD86, ICOSL, PD-L1 and PD-L2) that have one IgV domain and one Ig-C domain.
  • VISTA has one Ig-V domain like the receptors in the family. Accordingly, VISTA may also have a cell signaling function in addition to its ligand-like activity (of suppressing T cell activation).
  • V!STA is a novel and structurally-distinct, Ig-superfamily inhibitory ligand whose key features are:
  • VISTA-lg suppresses expression of early T cell activation antigens (CD69, CD44) as well as p-ERK activation suggesting that the receptor for VISTA (V-R) is expressed on resting T cells.
  • aVISTA mAb 13F3 exacerbated autoimmunity (EAE) (15).
  • aVISTA mAb induces tumor remission in multiple murine tumor models (17).
  • VISTA is constitutively expressed at high levels on myeloid cells (15, 16), and can suppress the activation of resting T cells, overtly activated T cells, as well as memory T cells (15, 16).
  • the inventors further theorized that the VISTA receptor would likely be expressed on resting T cells.
  • This theory was in part based on the fact that TCR-induced phospho-ERK activation (at 10 minutes post activation) can be completely arrested by VISTA-lg (Fig 1 ).
  • the earliest changes in T cell activation CD69, CD62L, CD44
  • VISTA-lg 1 15, 16).
  • VISTA-lg does not bind to any of the known B7 or PD family members (data not shown) or to cells that overexpress CD28, CTLA-4, PD-1. TLT- 2, TLT4, CD300A, and CD300D (unpublished data). Expression of VISTA-R Is predicted on resting T cells based on the ability of VISTA- lg to suppress aCD3 phospho-ERK signals at 10 minutes post-stimulation (Fig 1 ).
  • the inventors further predicted that the VISTA receptor will likely have no effect on B cell activation or proliferation and will likely not be appreciably expressed on resting or activated B cells.
  • VISTA as a negative regulator .
  • VISTA appears to negatively regulate (at least) tonic signaling of TCR (prior to cognate activation; Figure 2), serving a homeostatic function as a rheostat to prevent promiscuous T cell activation. This would agree with the high frequencies of activated T cells seen in the VISTA-/- mice (data not shown).
  • VISTA can also suppress T cell function post-activation, suggests that it may be an effective negative regulator after cognate activation of T cells as well.
  • PD-L1/PD-1 and CTLA-4/CD80/86 are inherently different pathways where receptors are only upregulated after T cell activation.
  • VISTA is exclusively hematopoietically-restricted , and therefore its major role would seem to be played in secondary lymphoid organs (SLO), or in sites where there is heavy leukocyte infiltration (like TME), unlike PD-L1 that is expressed in tissue on non- hematopoietic cells. Therefore, compounds which modulate VSIG8 or VISTA and/or the VISTA/VISTA-R interaction should have a fundamentally different role in controlling early T cell activities.
  • VISTA as a negative checkpoint regulator in cancer.
  • VISTA myeloid derived suppressor cells
  • TME tumor microenvironment
  • aVISTA treatment of mice in four different tumor models dramatically impairs tumor growth (Fig 3).
  • NCRs play a critical role in controlling the development of autoimmunity.
  • CTLA-4-/- mice develop aggressive systemic autoimmune disease (4-6), and PD-1-/- mice on a Balb/c bkg develop cardiomyopathy and mild lupus (7,19)
  • a major immune related toxicity in blocking PD-1 or CTLA-4 is the emergence of autoimmune disorders.
  • many NCRs impact on Treg function through an effect on natural (n)Treg or via enhancing the conversion of naive T cells to adaptive (a)Treg.
  • Ig fusion proteins of NCR ligands (B7-lgs) (B7-H4-lg, VISTA-lg, PD-L1-lg) that actively induce immunosuppressive signals through their respective receptors appear as effective therapeutics in the treatment of autoimmunity.
  • VISTA-lg can completely arrest aCD3-induced proliferation of T cells in vitro and a shutdown of virtually all early T cell activities (15- 17). Given that VISTA-lg can impair T cell activation, we assessed its therapeutic efficacy in a murine model of systemic lupus erythematosus.
  • One of the prevailing mechanisms underlying the progression of SLE nephritis implicates chronic activation of T cells with the subsequent recruitment of inflammatory myeloid cells into the kidney that facilitate pathology.
  • BWF1 The well-characterized mouse strain, BWF1 , develop an autoimmune renal disease similar to that seen in human lupus, including the presence of circulating self-reactive antibodies, increased pro-inflammatory cytokines, immune complex deposition glomerulonephritis (GN), progressive proteinuria, and ultimately death due to renal failure.
  • VISTA-lg immune complex deposition glomerulonephritis
  • Fig. 4 We have also shown a reduction in the frequency of activated T cells, an increase in Foxp3 signal and cytokine production in vivo with VISTA-lg treatment (data not shown).
  • V-R active signaling via V-R induces T ceil unresponsiveness and/or the emergence of aTreg (15, 16) depending on the strength of signal through the V-R.
  • VISTA-lg Similar therapeutic efficacy of VISTA-lg has also been shown in a murine model of multiple sclerosis, a conventional T cell mediated disease (not shown).
  • VISTA receptor Critical to the identification of the VISTA receptor is the production of high quality, multimeric ligands. The reason for this is that the predicted interaction of VISTA with its receptor is likely very low (uM). This prediction is based on the fact that the interaction between PD-1 and PD-L1 is low (Kd 526nM; as is PD-L2 (Kd 89nM). One can observe the PD-1 ->PD-L1 interaction if either the ligand or receptor is overexpressed (34). However, to otherwise observe PD-1 H»PD-L1 binding, one must use a multimeric ligand, like a tetramer. Alternatively, cells that are overexpressing ligand or receptor can be readily panned on plastic dishes coated with the complementary binding protein (35).
  • VISTA ligands suitable for dimerization, oligomerization or multimerization and for panning.
  • VISTA-lg has been engineered with N-terminal BirA sites for production of mu!timers with high molecular weight backbones.
  • ImmuDex ImmuDex
  • VISTA-dextramer VISTA-lg
  • VISTA-dextramer very high MW multimers that are created by attachment of ligands to a dextran backbone at pre-specified stoichiometries. These multimers are far more sensitive at detection of receptors than using conventional avidin-based tetramers. Preliminary data shows that VISTA-dextramer has activity of suppressing T cells in vitro (data not shown).
  • VISTA dimers may be used for V-R identification.
  • the original structural description of VISTA was modeled using structural motifs in the PD-L1 molecule(15) .
  • an alternative model based on a Fab fragment suggests that VISTA may form a dimer involving a disulphide bridge using a cysteine residue.
  • three glycosylation sites were located in the VISTA polypeptide which appear to be located a sufficient distance from the putative dimer interface site to allow dimer formation.
  • this fusion protein contains interchain disulphide bridges in the Fc part which may permit dimerization.
  • a relatively long and flexible linker results in a VISTA Ig fusion protein much more potent in activity than a fusin lacking such a long, flexible linker.
  • VISTA-CH3 may be a preferred ligand compared to VISTA-lg for V-R detection. Therefore, VISTA-CH3 with a C-terminal BirA site was envisioned to be suitable for use in enzymatic biotinylation for receptor discovery efforts. These VISTA !igands have not thus far been demonstrated to detectably bind to T cells. However, this is not surprising, due to the low affinity of the interactions of ligand and receptors in this family. As with other PD ligands, it was anticipated that these VISTA ligands when overexpressed would detectably bind V-R.
  • the inventors focused their efforts on V-R identification when using soluble ligands, wherein the soluble ligand used in the assay comprises a VISTA-dextramer (VISTA19-dex: 19 molecules of VISTA on a 250K dextran backbone) or comprises a VISTA dimer.
  • VISTA19-dex 19 molecules of VISTA on a 250K dextran backbone
  • plasmids contain both an open reading frame for the candidate gene in question, as well as GFP to confirm transfection and expression of the plasmid.
  • POC PD-1/GFP was transfected into s-CHO cells. After overnight culture, the specific adherence of GFP* cells can be easily observed and quantified to PD-L1 and not other plate-bound ligands (Fig 5).
  • CHO-S cells (Life Technologies) are transfected in a 96-well format with CMV-expression vectors for GFP-tagged proteins (Origene) from a candidate list of >200 selected genes. 24-hours later, cells are divided onto plates coated with either VISTA-Fc fusion protein or irrelevant Fc fusion protein and cultured for 1 hour. Non-adherent cells will be harvested by gentle pipetting to a new 96-well plate. Adherent cells are released by trypsin-EDTA treatment and both populations are examined by flow cytometry. This technique shows very little background, and positives are clearly visible even with light microscopy. Based thereon, CHO-S cells which overexpress V-R should detectably bind to VISTA-ig coated plates.
  • Recombinant, fluorescent VISTA19-dex may be used to screen a membrane protein library, e.g., the (-3800 unique genes) created by Retrogenix (UK) http://www.retrogenix.com to identify putative binding partners in a simple high throughput binding assay. Genes are expressed in an array of HEK293 cells and fluorochrome coupled ligand is used for detection of cells expressing a binding partner. 4. Yeast two-hybrid screening.
  • yeast two hybrid system such as the system developed by Hybrigenics (France) may be used to identify the putative receptor of VISTA, (http://www.hybrigenics-services.com) using ViSTA-ECD bait and prey library constructed from activated and non activated healthy human
  • the coding sequence for the extracellular domain of VISTA (aa 33-195; GenBank accession number gi: 62339431 ) is PCR-amplified and cloned into pB29 as an N-termina! fusion to LexA (N-VISTA-LexA-C).
  • the construct is corroborated for correctness by sequencing the entire insert and is then used as a bait to screen a random-primed leucocytes and activated mononuclear ceils cDNA library constructed into pP6, pB29 and pP6 derived from the original pBTM116 (37, 38) and pGADGH (39) piasmids, respectively.
  • 63 million clones (6-fo!d the complexity of the library) may be screened using a mating approach with YHGX13 (Y187 ade2-101 ::loxP-kanMX-loxP, mata) and L40aGal4 (mata) yeast strains as previously described (40). 161 His+ colonies are selected on a medium lacking tryptophan, leucine and histidine. The prey fragments of the positive clones are amplified by PGR and sequenced at their 5' and 3" junctions. The resulting sequences are used to identify the corresponding interacting proteins in the GenBank database (NCBI) using a fully automated procedure. A confidence score (PBS, for Predicted Biological Score) is then attributed to each interaction as previously described (41).
  • NBI GenBank database
  • VISTA-lg can suppress aCD3 activation of primary human T cells.
  • One of the limitations to only having an activity in primary cells is that genetic manipulation of primary cells is difficult.
  • Preliminary data now establishes that the upregulation of CD69 in Jurkat T cells, a transformed human T cell line, can be prevented using VISTA-lg (Fig 5). This finding allows the use of Jurkat cells to attempt to genetically silence the suppression of CD69 expression induced by VISTA to identify the V-R.
  • the Mission shRNA whole genome library from Sigma is used. The library is divided into 10 tubes, each tube containing -12,000 shRNA constructs targeting ⁇ 2,000 genes.
  • the Jurkat cells are transfected with the library at a MOI such that only one gene is knocked down per cell.
  • the transduced cells wiii be cultured in the presence of Puromycin and/or sorted to select shRNA expressing cells.
  • the stably knocked down cells will be stimulated with anti-CD3+/- VISTA-fg for 24 hrs and the CD69 + cells in the presence of VISTA-lg will be counted as positive hits.
  • the restoration of IL- 2 production will also be used to address receptor silencing.
  • the CD69+ cells will be sorted and the identity of shRNA will be carried out by Sigma using deep sequencing and deconvolution techniques.
  • the specificity of top candidates will be tested by single gene knockdown and over expression approaches in both the Jurkat cells and primary T cells using additional assay e.g. proliferation and multiple cytokines.
  • the resulting cDNA library will be directly swapped into a Gateway® compatible DEST expression vector downstream of a human elongation factor 1 -alpha promoter and upstream of an IRES (internal ribosome entry site)-ZsGreen1 fluorescent protein cassette to track co-expression of cloned cDNA inserts from the same mRNA species.
  • Serial rounds of panning of libra ry-transfected CHO-S cells on VISTA-lg coated plates will be performed to enrich for VISTA receptor expression. When a high frequency of cells adhere to plates (>50%), plasmid will be isolated, transformed into bacteria for clonal expansion and sequenced.
  • a putative VISTA-R candidate identified by any of the afore-described methods may be evaluated in functional assays to confirm that the binding
  • Blocking of VISTA function by anti - V-R monoclonal antibody Reversal of VISTA-lg mediated T cell suppression in the presence of antl-V-R monoclonal antibody
  • Recombinant VISTA-lg fusion protein suppresses anti-CD3 (OKT3) mediated in vitro T ceil proliferation and cytokine production in a dose dependent manner. Therefore, in the presence of anti-V-R antibody (which is a
  • 96 well plates are coated with either anti-CD3 antibody (OKT3) alone or anti-CD3 plus VISTA-lg at 37 °C for 1 hr.
  • the anti-CD3 and VISTA-lg may be used at a final concentration of 2.5 and 10 ug/ml respectively.
  • Two hundred thousand purified human T cells from healthy donor peripheral blood are added to each well. The duplicate experiment will be carried out in the presence of anti-V-R mAb. The plates are incubated at 37 °C for 5 days.
  • V-R The effects of V-R on VISTA function may be further corroborated using T or Jurkat cell lines wherein the expression of the putative V-R is knocked out.
  • the recombinant ViSTA-lg fusion protein suppresses anti-CD3 (OKT3) induced CD69 and IL-2 upregulation in Jurkat cells, a lymphoma cell line, in a dose dependent manner.
  • the VISTA receptor candidate is knocked down in Jurkat cells using lentivirus shRNA silencing technique. Such knocked-out cells will be
  • the gene specific shRNA lentiviral particles are constructed to knock down the VSIG8 (Mission shRNA particles, TCR library from Sigma).
  • the Jurkat cells will be transduced with shRNA lentiviral particles at an optimized multiplicity of infection to achieve greater than 90% knock down of target gene without nonspecific gene deletion.
  • the transduced cells are selected in the presence of puromycin drug.
  • the specificity and the level of knockdown of the target gene is confirmed by quantitative PGR.
  • the cells will be stimulated with anti-CD3 in the presence or absence of VISTA-lg.
  • the level of CD69 will be assessed by flow cytometry and IL-2 will be measured by ELISA in the culture supernatant after 72 hrs.
  • the recombinant VISTA-lg fusion protein suppresses anti-CD3 (OKT3) mediated in vitro T cell proliferation and cytokine production in a dose dependent manner.
  • the VISTA receptor candidate is knocked down in T cells using lentivirus shRNA silencing technique. Such knocked-out cells will should be unresponsive to VISTA-lg mediated suppression.
  • the gene specific shRNA lentiviral particles are constructed to knock down the VSIG8 (Mission shRNA particles, TCR library from Sigma).
  • the T cells from healthy donor will be transduced with shRNA lentiviral particles at an optimized multiplicity of infection to achieve greater than 90% knock down of target gene without nonspecific gene deletion.
  • the transduced cells will be selected in the presence of puromycin drug.
  • the specificity and the level of knockdown of the target gene will be confirmed by quantitative PGR.
  • the anti-CD3 mediated T cell proliferation assay will be performed as described above in the presence or absence of VISTA-lg. The cytokine and cell proliferation will be assessed.
  • the confirmation of a putative V-R as being the authentic receptor for VISTA may be further corroborated by detecting mRNA and protein expression on T cells.
  • the VISTA receptor was elucidated and found to be V-set and immunoglobulin domain-containing protein 8 or VSIG8.
  • the identified V-R is used to identify VISTA/V-R agonists or antagonists.
  • agonist and antagonist compounds will include in particular antibodies and antibody fragments that agonize or antagonize the effects of VSIG8 on T or NK immunity and preferably those which inhibit or promote the interaction of VISTA and
  • VSIG8 antibodies may be obtained by well-known in vivo or in vitro immunization using the VSIG8 polypeptide or a fragment or conjugate thereof as an immunogen.
  • these antibodies and antibody fragments will include human, humanized, primatized and chimeric antibodies an antibody fragments such as Fab, Fab', scFv, (Fabfe, IgNars, metMabs, and other known types of antibodies and antibody fragments.
  • These agonist and antagonist compounds will further include polypeptides, i.e., polypeptides which comprise all or a portion of the extracellular region of VSIG8 or a polypeptide which possesses at least 80, 90, 95 or 99% sequence identity to the extracellular region of VSIG8 or a portion thereof such as an IgV or IgC domain therein.
  • polypeptides i.e., polypeptides which comprise all or a portion of the extracellular region of VSIG8 or a polypeptide which possesses at least 80, 90, 95 or 99% sequence identity to the extracellular region of VSIG8 or a portion thereof such as an IgV or IgC domain therein.
  • These VSIG8 polypeptides may also be fused to another polypeptide such as an Ig constant region, e.g., an lgG1 , lgG2, lgG3 or lgG4 constant region which optionally may be mutagenized to enhance or inhibit FcR and/or complement
  • the agonist or antagonist may comprise one or more copies of the VSIG8 polypeptide or fragment, i.e., the compound may be multimeric and the copies of the VSIG8 polypeptide may be intervened by a linker, e.g., a long, flexible peptide such as one which is at least 15-25 amino acids and containing one or more serine residues.
  • a linker e.g., a long, flexible peptide such as one which is at least 15-25 amino acids and containing one or more serine residues.
  • agonist and antagonist compounds according to the invention will further include small molecules which agonize or antagonize the VISTA/VSIG8 binding interaction.
  • an antagonist according to the invention will substantially inhibit or prevent the suppressive effects of VISTA on T or NK cell immunity. This may be detected using in vitro cell based assays with cells that express VISTA and/or VSIG8.
  • An antagonist will inhibit or block the suppressive effects mediated by VISTA on T cell activation or proliferation or NK T cell activation or proliferation, e.g., it will increase CD8 + or CD4 + T cell activation or CDS* or CD4 + T cell proliferation and/or promote NK cell mediated activities compared to suitable controls.
  • an agonist according to the invention preferably will potentiate or enhance the suppressive effects of VISTA on T or NK cell immunity. This may be detected using in vitro using cell based assays with cells that express VISTA and/or VSIG-8.
  • An agonist will promote the suppressive effects mediated by VISTA on T cell activation or proliferation or on NK cell mediated activities, e.g., it will suppress CDS* or CD4* T cell activation or proliferation or NK mediated cellular functions such as lysis compared to suitable controls.
  • the screening methods used to identify such agonists and antagonists may be affected in high throughput format if desired.
  • the identified agonists and antagonists may be formulated for use in human therapy. This may comprise the addition of suitable stabilizers, excipients or carriers.
  • the agonist or antagonist may be modified to enhance in vivo stability such as by the attachment to one or more water-soluble polymers such as polyethylene glycol polymers or by acylation. Methods for attaching such moieties to proteins are well known in the art.
  • compositions containing agonists according to the invention may be used to inhibit T cell immunity and to treat conditions where this is
  • compositions will comprise an amount of an agonist according to the invention effective to suppress T cell activation or proliferation in a subject in need thereof.
  • autoimmune, inflammatory and allergic conditions include for example arthritic conditions such as RA, psoriatic arthritis, scleroderma, multiple sclerosis, lupus, IBD, ITP, diabetes, sarcoidosis, allergic asthma, and the like.
  • compositions containing antagonists according to the invention may be used to promote T ceil immunity and to treat conditions where this is therapeutically desirable such as cancer and infectious disease indications. These compositions will comprise an amount of an antagonist according to the invention effective to promote T cell activation or proliferation in a subject in need thereof, e.g. a subject with a cancerous condition.
  • Cancers treatable with the subject antagonists include any way of example melanoma, lymphoma, leukemia, lung cancer, ovarian cancer, cervical cancer, testicular cancer, digestive cancers, esophageal cancer, liver cancers, pancreatic cancer, kidney cancer, skin cancer.
  • Infectious diseases treatable with antagonists according to the invention include viral diseases such as HIV, HPV, EBV, encephalitis, herpes, other pox viruses, and other known human viruses, parasitic diseases, bacterial diseases, fungal or yeast associated diseases, as well as other infectious disease conditions that affect humans.
  • viral diseases such as HIV, HPV, EBV, encephalitis, herpes, other pox viruses, and other known human viruses, parasitic diseases, bacterial diseases, fungal or yeast associated diseases, as well as other infectious disease conditions that affect humans.
  • the subject agonists or antagonists may be combined with other therapeutics which may be administered in the same or different compositions, at the same or different time.
  • the subject agonists or antagonists may be administered in a therapeutic regimen that includes the administration ofd a PD-1 or PD-L1 agonist or antagonist, CTLA4-lg, a cytokine, a cytokine agonist or antagonist, or another receptor agonist or antagonist.
  • an anti- VSIG8 antibody according to the
  • such anti- VSIG8 antibody may comprise or consist of a human antibody comprising heavy or light chain variable regions that are "the product of or "derived from” a particular germline sequence if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin genes.
  • Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest.
  • a human antibody that is "the product of or "derived from” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody.
  • a human antibody that is "the product of or "derived from” a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally-occurring somatic mutations or intentional introduction of site-directed mutation.
  • a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline
  • a human antibody may be at least 95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.
  • a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene.
  • the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
  • an anti- VSIG8 antibody according to the
  • the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to isolated anti- VSIG8 amino acid sequences of preferred anti- VSIG8antibodies, respectively, wherein the antibodies retain the desired functional properties of the parent anti- VSIG8 antibodies.
  • percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available commercially), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • an anti- VSIG8 antibody according to the
  • inventions comprises a heavy chain variable region comprising CDRI, CDR2 and CDR3 sequences and a light chain variable region comprising CDRI, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specified amino acid sequences based on preferred anti- anti-VSIG8 antibodies isolated and produced using methods herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of anti- VSIG8 antibodies according to at least some embodiments of the invention, respectively.
  • the anti-VSIG8 antibody can be, for example, human antibodies, humanized antibodies or chimeric antibodies.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding
  • Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,
  • one or more amino acid residues within the CDR regions of an antibody according to at least some embodiments of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through j) above) using the functional assays described herein.
  • an anti- VSIG8 antibody according to the
  • a desired functional property such as modulation of immune stimulation and related functions.
  • Other antibodies with the same epitope specificity may be selected and will have the ability to cross-compete for binding to VSIG8 antigen with the desired antibodies.
  • the epitopic specificity of a desired antibody may be determined using a library of
  • overlapping peptides comprising the entire VSIG8 polypeptide, e.g., 15-mers or an overlapping peptide library constituting a portion containing a desired epitope of VSIG8 and antibodies which bind to the same peptides or one or more residues thereof in the library are determined to bind the same linear or conformational epitope.
  • an anti- VSIG8 antibody according to the
  • VH and/or VL sequences derived from an anti- VSIG8 antibody starting material can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant regions, for example to alter the effector functions of the antibody.
  • CDR grafting One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody- antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al.
  • Suitable framework sequences can be obtained from public DNA
  • germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet), as well as in Kabat, E. A., et al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.
  • variable region modification is to mutate amino acid
  • Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications (as discussed above) are introduced.
  • the mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.
  • one approach is to "backmutate" one or more framework residues to the corresponding germline sequence.
  • an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • antibodies according to at least some embodiments of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen- dependent cellular cytotoxicity.
  • an antibody according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Such embodiments are described further below.
  • the numbering of residues in the Fc region is that of the EU index of Kabat.
  • the hinge region of CHI is modified such that the
  • CHI number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc -hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc -hinge domain SpA binding.
  • SpA Staphylococcal protein A
  • the antibody is modified to increase its biological half-life.
  • Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121 ,022 by Presta et al.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector figand but retains the antigen-binding ability of the parent antibody.
  • the effector !igand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301 , 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331 , 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439, This approach is described further in PCT Publication
  • the antibody can be modified to abrogate in vivo Fab arm exchange.
  • th s process involves the exchange of igG4 half-molecules (one heavy chain plus one light chain) between other lgG4 antibodies that effectively results in b specific antibodies which are functionally monovalent. Mutations to the hinge region and constant domains of the heavy chain can abrogate this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19).
  • the glycosylation of an antibody is modified.
  • an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
  • Such carbohydrate modifications can be
  • aglyciosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosy! residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host ceil with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express
  • the ceil ilnes Ms7G4, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (a (1 ,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 eel! lines lack fucose on their carbohydrates.
  • the Ms704, Ms705, and Ms709 FUT8 ceil lines are created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004)
  • EP 1 ,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the a 1 ,6 bond-related enzyme.
  • Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662).
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to
  • the fucose residues of the antibody may be cleaved off using a fucosidase enzyme.
  • the fucosidase a-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).
  • Another modification of the antibodies herein that is contemplated by the invention is pegyiation or the addition of other water soluble moieties, typically polymers, e.g., in order to enhance half-life.
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • PEG polyethylene glycol
  • the pegyiation is carried out via an acyiation reaction or an aikyiation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • PEG polyethylene glycol
  • the pegyiation is carried out via an acyiation reaction or an aikyiation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Ci-Cio) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody.
  • an anti- VSIG8 antibody according to the
  • VH and V L sequences can be used to create new anti- VSIG8 antibodies, respectively, by modifying the VH and/or V
  • the structural features of an anti- VSIG8 antibody according to at least some embodiments of the invention are used to create structurally related anti- VSIG8 antibodies that retain at least one functional property of the antibodies according to at least some embodiments of the invention, such as binding to human VSIG8.
  • one or more CDR regions of one VSIG8 antibody or mutations thereof can be combined
  • the starting material for the engineering method is one or more of the V H and/or V L sequences provided herein, or one or more CDR regions thereof.
  • To create the engineered antibody it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequences is used as the starting material to create a "second generation" sequences derived from the original sequences and then the "second
  • the anti- VSIG8 antibody encoded by the altered antibody sequences is one that retains one, some or all of the functional properties of the anti- VSIG8 antibodies, respectively, produced by methods and with sequences provided herein, which functional properties include binding to VSIG8 antigen with a specific KD level or less and/or modulating immune responses and/or selectively binding to desired target cells such as for example, that express VSIG8 antigen.
  • the functional properties of the altered antibodies can be assessed using standard assays available in the art and/or described herein.
  • mutations can be introduced randomly or selectively along all or part of an anti- VSIG8 antibody coding sequence and the resulting modified anti- VSIG8 antibodies can be screened for binding activity and/or other desired functional properties.
  • soluble ectodomain or "ectodomain” or “soluble” form of VSIG8 refers also to the nucleic acid sequences encoding the
  • the VSIG8 ECD proteins and fragments thereof refer to any one of the polypeptide sequences listed in any of SEQ ID NOs: 1-3, and/or variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith, and/or fusions and or conjugates thereof, and/or polynucleotides encoding same.
  • the fragments of the extracellular domain of VSIG8 can include any sequence corresponding to any portion of or comprising the IgV domain of the extracellular domain of VSIG8.
  • the VSIG8 proteins contain an immunoglobulin domain within the
  • the IgV domain (or V domain), which is related to the variable domain of antibodies.
  • the IgV domain may be responsible for receptor binding, by analogy to the other B7 family members.
  • the Ig domain of the extracellular domain includes one disulfide bond formed between intra domain cysteine residues, as is typical for this fold and may be important for structure- function.
  • a soluble fragment of VSIG8 as described in greater detail below with regard to the section on fusion proteins, such a soluble fragment may optionally be described as a first fusion partner.
  • Useful fragments are those that alone or when comprised in fusion proteins or multimerized retain the ability to bind to their natural receptor or receptors, e.g., expressed on T and NK cells, and/or which modulate (inhibit or promote) T cell and/or NK cell activation.
  • a VSIG8 polypeptide that is a fragment of full-length VSIG8 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural receptors) and/or the modulation (agonism or antagonism) of one or more of the functional effects of VSIG8 on immunity and on specific immune cells as compared to full-length VSIG8.
  • Soluble VSIG8 polypeptide fragments are fragments of VSIG8 polypeptides that may be shed, secreted or otherwise extracted from the producing cells.
  • the soluble fragments of VSIG8 polypeptides include fragments of the VSIG8 extracellular domain that retain VSIG8 biological activity, such as fragments that retain the ability to bind to their natural receptor or receptors and/or which modulate (inhibit or promote) T or NK cell activation.
  • the extracellular domain can include 1 , 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1 , 2, 3, 4, or 5 contiguous amino acids from the signal sequence.
  • the extracellular domain can have 1 , 2, 3, 4, 5 or more
  • the VSIG8 extracellular domain polypeptide comprises the amino acid sequence of the IgV domain as set forth in any one of SEQ ID NO: 1 , 2 or 3, or fragments or variants thereof. In other embodiments the VSIG8 extracellular domain polypeptide consists essentially of the amino acid sequence of the IgV domain as set forth in any one of SEQ ID NOs: 1-3.
  • the VSIG8 polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence.
  • the signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence.
  • the signal sequence of VSIG8 can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide.
  • the signal peptide sequence that is used to replace the VSIG8 signal peptide sequence can be any known in the art.
  • soluble ectodomain (ECD)" or “ectodomain” or “soluble” form of VSIG8 will modulate (agonize or antagonize) one or more of VSIG8's effects on immunity and specific types of immune cells such as cytotoxic or effector T cells, Tregs and NK cells.
  • the present invention encompasses useful variants of VSIG8 polypeptides including those that increase biological activity, as indicated by any of the assays described herein, or that increase half-life or stability of the protein.
  • Soluble VSIG8 proteins or fragments, or fusions thereof having VSIG8 protein activity, respectively can be engineered to increase biological activity.
  • the VSIG8 protein or fusion protein is modified with at least one amino acid substitution, deletion, or insertion that increases the binding of the molecule to an immune cell, for example a T cell, and transmits an inhibitory signal into the T cell.
  • An isolated or recombinant VSIG8 polypeptide or fusion protein according to any of the foregoing claims which comprises at least one half-life extending moiety.
  • Such half-life extending moieties may include by way of example
  • PEG polyethylene glycol
  • mPEG monomethoxy PEG
  • XTEN XTEN
  • rPEG rPEG molecule
  • adnectin a serum albumin, human serum albumin, immunoglobulin constant region or fragment thereof, or acy! group.
  • the half-life modified isolated or recombinant VSIG8 polypeptide or fusion protein according to the invention which comprises a heterologous polypeptide, or half-life extending moiety, or other heterologous molecule may increase the in vivo half-life of the VSIG8 polypeptide or fusion protein by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5- fold, at least about 10-fold, or more compared to an otherwise identical molecule that lacks said heterologous polypeptide, half-life extending moiety, or other heterologous molecule.
  • VSIG8 proteins that are engineered to selectively bind to one type of T cell versus other immune cells or to NK cells.
  • the VSIG8 polypeptide can be engineered to bind optionally to Tregs, ThO, Th1 , Thl7, Th2 or Th22 cells or to NK cells.
  • Preferential binding refers to binding that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater for one type of cell over another type of cell.
  • Still other variants of VSIG8 protein can be engineered to have reduced binding to immune cells relative to wild-type VSIG8 protein, respectively. These variants can be used in combination with variants having stronger binding properties to modulate the immune response with a moderate impact.
  • variant VSIG8 protein can be engineered to have an
  • variants typically are modified to resist enzymatic degradation.
  • exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation.
  • Various modifications to achieve this are known in the art.
  • VSIG8 antagonize
  • VSIG8's effects on immunity and on specific types of immune cells such as cytotoxic or effector T cells, Tregs, MDSCs, other suppressor cell types or on NK cells.
  • VSIG8 fusion polypeptides have a first fusion partner comprising all or a part of a VSIG8 protein fused to a second polypeptide directly or via a linker peptide sequence or a chemical linker useful to connect the two proteins.
  • the VSIG8 polypeptide may optionally be fused to a second polypeptide to form a fusion protein as described herein.
  • the presence of the second polypeptide can alter the solubility, stability, affinity and/or valency of the VSIG8 fusion polypeptide.
  • "valency" refers to the number of binding sites available per molecule.
  • the second polypeptide is a polypeptide from a different source or different protein.
  • the VSIG8 protein or fragment is selected for its activity for the treatment of immune related disorder, infectious disorder, sepsis, cancer, and/or for blocking the undesirable immune activation that follows gene transfer, as described herein.
  • the second polypeptide contains one or more
  • the fusion protein is a dimeric fusion protein which optionally is capable of cross-linking two or more targets.
  • the dimer results from the covalent bonding of Cys residue in the hinge region of two of the Ig heavy chains that are the same Cys residues that are disulfide linked in dimerized normal ig heavy chains.
  • Such proteins are referred to as VSIG8 polypeptides, fragments or fusion proteins thereof.
  • the immunoglobulin constant domain may contain one or more amino acid insertions, deletions or substitutions that enhance or decrease binding to specific cell types, increase the bioavailability, or increase the stability of the VSIG8 polypeptides, fusion proteins, or fragments thereof.
  • Suitable amino acid substitutions include conservative and non-conservative substitutions, as described above.
  • the fusion proteins optionally contain a domain that functions to dimerize or multimerize two or more fusion proteins.
  • the peptide/polypeptide linker domain can either be a separate domain, or alternatively can be contained within one of the other domains (VSIG8 polypeptide or second polypeptide) of the fusion protein.
  • the domain that functions to dimerize or multimerize the fusion proteins can either be a separate domain, or alternatively can be contained within one of the other domains (VSIG8 polypeptide, second polypeptide or peptide/polypeptide linker domain) of the fusion protein.
  • peptide/polypeptide linker domain are the same. Further specific, illustrative and non-limiting examples of dimerization/multimerization domains and linkers are given below.
  • Fusion proteins disclosed herein are of formula I: N-R1-R2-R3-C wherein "N” represents the N-terminus of the fusion protein, “C” represents the C-terminus of the fusion protein.
  • Rl is a VSIG8 polypeptide
  • R2 is an optional peptide/polypeptide or chemical linker domain
  • R3 is a second polypeptide.
  • R3 may be a VSIG8 polypeptide and Rl may be a second polypeptide.
  • linkers are described in greater detail below.
  • the fusion protein comprises the VSIG8 polypeptide fragments as described herein, fused, optionally by a linker peptide of one or more amino acids (e.g. GS) to one or more "half-life extending moieties".
  • a "half-life extending moiety” is any moiety, for example, a polypeptide, small molecule or polymer, that, when appended to protein, extends the in vivo half-life of that protein in the body of a subject (e.g., in the plasma of the subject).
  • a half-life extending moiety is, in an embodiment of the invention, polyethylene glycol (PEG), monomethoxy PEG (mPEG), XTEN molecule, an rPEG molecule, an adnectin, a serum albumin, human serum albumin, immunoglobulin constant region or fragment thereof, or acyl group.
  • PEG is a 5, 10, 12, 20, 30, 40 or 50 kDa moiety or larger or comprises about 12000 ethylene glycol units (PEG12000).
  • An isolated or recombinant VSIG8 polypeptide or fusion protein according to the invention may optionally comprise at least one half-life extending moiety.
  • Half-life extending moieties include PEG's, an XTEN molecule, an rPEG molecule, an adnectin, a serum albumin, human serum albumin, immunoglobulin constant region or fragment thereof, or acyl group.
  • the heterologous polypeptide, half-life extending moiety, or other heterologous molecule contained in a VSIG8 polypeptide or fusion protein according to the invention may increase the in vivo half-life of said isolated or recombinant VSIG8 polypeptide or fusion protein by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5- fold, at least about 10-fold, or more compared to an otherwise identical molecule that lacks said heterologous polypeptide, half-life extending moiety, or other heterologous molecule.
  • the fusion protein may also optionally be prepared by chemical synthetic methods and the "join" effected chemically, either during synthesis or post- synthesis.
  • Cross-linking and other such methods may optionally be used (optionally also with the above described genetic level fusion methods), as described for example in US Patent No. 5,547,853 to Waliner et al, which is hereby
  • a fusion protein may be prepared from a protein of the invention by fusion with a portion of an immunoglobulin comprising a constant region of an immunoglobulin. More preferably, the portion of the immunoglobulin comprises a heavy chain constant region which is optionally and more preferably a human heavy chain constant region.
  • the heavy chain constant region is most preferably an IgG heavy chain constant region, and optionally and most preferably is an Fc chain, most preferably an IgG F c fragment that comprises the hinge, Cm and Cm domains.
  • the Fc chain may optionally be a known or "wild type" Fc chain, or alternatively may be mutated or truncated.
  • the F c portion of the fusion protein may optionally be varied by isotype or subclass, may be a chimeric or hybrid, and/or may be modified, for example to improve effector functions, control of half-life, tissue accessibility, augment biophysical characteristics such as stability, and improve efficiency of production (and less costly).
  • Many modifications useful in construction of disclosed fusion proteins and methods for making them are known in the art, see for example Mueller, et al, Moi. Immun., 34(6):441-452 (1997), Swann, et al., Curr. Qpin. immun.. 20:493-499 (2008), and Presta, Curr. Opin. Immun. 20:460- 470 (2008).
  • the Fc region is the native IgGI, lgG2, lgG3 or lgG4 Fc region. In some embodiments the F c region is a hybrid, for example a chimeric consisting of lgG2/lgG4 Fc constant regions.
  • Modifications to the Fc region include, but are not limited to, lgG4
  • the Fc region may include the entire hinge region, or less than the entire hinge region. In another embodiment, the Fc domain may contain one or more amino acid insertions, deletions or
  • Another embodiment includes igG2-4 hybrids and lgG4 mutants that have reduced binding to FcR (F c receptor) which increase their half-life.
  • IgGI and/or lgG2 domain is deleted; for example, Angal et al. Molecular Immunology, 30(1 ): 105-108 (1993) describe IgGI and lgG2 having serine 241 replaced with a proline.
  • the Fc domain contains amino acid insertions, deletions or substitutions that enhance binding to CD 16 A.
  • a large number of substitutions in the Fc domain of human IgGI that increase binding to CD16A and reduce binding to CD32B are known in the art and are described in
  • Exemplary variants of human IgGI Fc domains with reduced binding to CD32B and/or increased binding to CD16A contain F243L, R929P, Y300L, V305I or P296L substitutions. These amino acid substitutions may be present in a human IgGI Fc domain in any combination.
  • the human IgGI Fc domain variant contains a F243L, R929P and Y300L substitution. In another embodiment, the human IgGI Fc domain variant contains a F243L, R929P, Y300L, V305I and P296L substitution. In another embodiment, the human IgGI Fc domain variant contains an N297A/Q substitution, as these mutations abolish FcyR binding.
  • Non-limiting, illustrative, exemplary types of mutations are described in US Patent Application No.
  • Fc chain also optionally comprises any type of Fc fragment.
  • substitution or exclusion of these specific amino acids therefore allows for inclusion or exclusion of specific immunoglobulin constant region-mediated activity.
  • specific changes may result in aglycosylation for example and/or other desired changes to the Fc chain. At least some changes may optionally be made to block a function of Fc which is considered to be
  • Non-limiting, illustrative examples of mutations to Fc which may be made to modulate the activity of the fusion protein include the following changes (given with regard to the Fc sequence nomenclature as given by Kabat, from Kabat EA et al: Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, (1991 )): 220C - > S; 233-238 ELLGGP - > EAEGAP; 265D -> A, preferably in combination with 434N -> A; 297N - > A (for example to block N-glycosylation); 318-322 EYKCK - > AYACA; 330-331 AP - > SS; or a combination thereof (see for example M. Clark, Chemical Immunol and Antibody Engineering, pp 1-31 for a description of these mutations and their effect).
  • the construct for the Fc chain which features the above changes optionally and preferably comprises a combination of the hinge region with the Cm and Cm
  • the above mutations may optionally be implemented to enhance desired properties or alternatively to block non-desired properties.
  • aglycosylation of antibodies was shown to maintain the desired binding functionality while blocking depletion of T-cells or triggering cytokine release, which may optionally be undesired functions (see M. Clark, Chemical Immunol and Antibody Engineering, pp 1-31 ).
  • Substitution of 331 proline for serine may block the ability to activate complement, which may optionally be considered an undesired function (see M. Clark, Chemical Immunol and Antibody Engineering, pp 1-31 ).
  • Changing the alanine to serine at position 330 in combination with this change may also enhance the desired effect of blocking the ability to activate complement.
  • Residues 235 and 237 were shown to be involved in antibody-dependent cell-mediated cytotoxicity (ADCC), such that changing the block of residues from 233-238 as described may also block such activity if ADCC is considered to be an undesirable function.
  • Residue 220 is normally a cysteine for F c from IgGI, which is the site at which the heavy chain forms a covalent linkage with the light chain.
  • this residue may be changed to another amino acid residue (e.g., serine), to avoid any type of covalent linkage (see M. Clark, Chemical Immunol and Antibody Engineering, pp 1-31 ) or by deletion or truncation.
  • the fusion protein includes the extracellular domain of VSIG8 or a fragment thereof fused to an Ig F c region.
  • Recombinant Ig VSIG8 polypeptides, fragments or fusion proteins thereof fusion proteins can be prepared by fusing the coding region of the extracellular domain of VSIG8 or a fragment thereof to the Fc region of human IgGI or mouse lgG2a, as described previously (Chapoval, et al., Methods Mol. Med, 45:247-255 (2000)).
  • VSIG8 ECD refers also to fusion protein, comprising an amino acid sequence of human VSIG8 ECD fused to human immunoglobulin F c .
  • said fusion protein comprises the amino acid sequence of the human or non-human VSIG8 ECD set forth in SEQ ID NOs: 1 , 2, 3 or fragment thereof
  • the second polypeptide may have a conjugation domain through which additional molecules can be bound to the VSIG8 fusion proteins.
  • the conjugated molecule is capable of targeting the fusion protein to a particular organ or tissue; further specific, illustrative, non-limiting examples of such targeting domains and/or molecules are given below.
  • conjugated molecule is another
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise a binding domain, wherein the binding protein is capable of cross-linking two or more targets.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise another binding moiety, wherein the binding moiety targets a tumor cell, infectious agent, e.g., a virus, bacterium, mycoplasm, fungus, yeast or parasite, or cell infected thereby, an immune cell, or a disease site.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one heterologous polypeptide which may be a receptor, hormone, cytokine, antigen, B-cell target, NK cell target, T cell target, TNF receptor superfamily member, Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF- ⁇ superfamily member, a Wnt-related molecule, a receptor ligand, a Dendritic ceil target, a myeloid cell target, a monocyte/macrophage cell target or an angiogenesis target.
  • heterologous polypeptide which may be a receptor, hormone, cytokine, antigen, B-cell target, NK cell target, T cell target, TNF receptor superfamily member, Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF- ⁇ superfamily member, a Wnt
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one antigen, e.g., a tumor antigen,
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise a T cell target selected from the group consisting of 2B4/SLAMF4, IL-2 Ra, 4-1 BB/TNFRSF9, IL-2R , ALCAM, B7-1/CD80, IL-4R, B7-H3, BLAME/SLAMF8, BTLA, IL-6R, CCR3, IL-7 Ra, CCR4, CXCRI/IL-8 RA, CCR5, CCR6, IL-10 R a, CCR7, IL-10 R , CCR8, !L-12 R i, CCR9, IL-12 R$2, CD2, IL-13Ral, IL-13, CDS, CD4, ILT2/CD85j, ILT3/CD85k, ILT4/CD85d,
  • ILT5/CD85a Integrin a 4/CD49d, CDS, lntegrinaE/CD103, CD6, Integrin a M/CD! !b, CD8, Integrin a X/CD 11c, Integrin 2/CD18, KIR/CD158,
  • CD27/TNFRSF7 CD27/TNFRSF7, KIR2DL1 , CD28, KIR2DL3, CD30/TNFRSF8,
  • KIR2DL4/CD158d CD31/PECAM-1 , KIR2DS4, CD40 Ligand/TNFSFS, LAG-3, CD43, LAIR1 , CD45, LAIR2, CD83, Leukotriene B4 Rl, CD84/SLAMF5, NCAM- Ll, CD94, NKG2A, CD97, NKG2C, CD229/SL AMF3 , NKG2D, CD2F- 10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common y Chain/IL-2 Ry,
  • RANK/TNFRSF11 A CX3CR1 , CX3CL1 , L-Selectin, CXCR3, SIRP pi, CXCR4, SLAM, CXCR6, TCCRAVSX-1 , DNAM-1 , Thymopoietin, EMMPRIN/CD 147, TIM-1 , EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fey RIII/CD16, TIM-6, GITR/TNFRSF18, TNF RI/TNFRSFIA, Granulysin, TNF R11/TNFRSF1B, H VEM/TNFRSF 14, TRAIL R1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-yRI, TRAIL R4/TNFRSF10D, IFN-yR2, TSLP, IL
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one monocyte/macrophage cell target selected from the group consisting of B7-1/CD80, ILT4/CD85d, B7-H1 ,
  • ILT5/CD85a Common ⁇ Chain
  • Integrin a 4/CD49d BLAME/SLAMF8, Integrin a X/CD1 lc, CCL6/C10, Integrin ⁇ 2/CD18, CD155/PVR, Integrin ⁇ 3/CD61 , CD31/PECAM-1 , Latexin, CD36/SR-B3, Leukotriene B4 Rl, CD40/TNFRSF5, LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c, CD68,
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one Dendritic cell target is selected from the group consisting of CD36/SR-B3, LOX-1/SR-E1 , CD68, MARCO, CD163, SR- AI/MSR, CD5L, SREC-I, CL-P 1/COLEC 12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLRI, TLR5, TLR2, TLR6, TLR3, TLR9, 4- IBB Ligand/TNFSF9, IL-12/IL- 23 p40, 4-Amino-l,8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8- oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, Integrin a 4/CD49d, Aag, Integrin ⁇ 2/CD18,
  • LMIR3/CD300LF CCR6, LMIR5/CD300LB, CCR7, LMIR6/CD300LE,
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one TNF receptor superfamily member is selected from the group consisting of 4-1BB/TNFRSF9, NGF R/TNFRSF16, BAFF R/TNFRSF13C, Osteoprotegerin/TNFRSFI IB, B CMA/TNFRSF 17 , OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF11 A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5 , T ACI/TNFRSF 13B , DcR3/TNFRSF6B , TNF RI/TNFRSF1A, DcTRAIL R1/TNFRSF23, TNF RII/TNFRSF1 B, DcTRAIL R2/TNFRSF22, TRAIL R1/TNFRSF10A, DR3/TNFRSF25 , TRAIL
  • R2/TNFRSF10B DR6/TNFRSF21 , TRAIL R3/TNFRSF10C, EDAR, TRAIL R4/TNFRSF 10D , Fas/TNFRSF6, TRO Y/TNFRSF 19 , GITR/TNFRSF18 , TWEAK R/TNFRSF12, HVEM/TNFRSF14, XEDAR, Lymphotoxin ⁇
  • Ligand/TNFSF7 S TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-a/TNFSFIA, CD40 Ligandn " NFSF5, TNF- /TNFSFIB, EDA-A2, TRAILJTNFSFIO, Fas
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one Hedgehog family member selected from the group consisting of Patched and Smoothened.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one receptor tyrosine kinase selected from the group consisting of Axl, FGF R4, Clq R1/CD93, FGF R5, DDR1 , Flt-3, DDR2, HGF R, Dtk, IGF-I R.
  • EGF R IGF-II R, Eph, INSRR, EphAI, Insulin R/CD220, EphA2, M-CSF R, EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R a, EphA7, PDGF R ⁇ , EphA8, Ret, EphBI, RORI, EphB2, ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1 , EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF Rl, VEGF RI/Flt-1 , FGF R2, VEGF R2/Flk-1 , FGF R3 and VEGF R3/Flt-4.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one Transforming Growth Factor (TGF)- superfamily member selected from the group consisting of Activin RIA/ALK-2, GFR a-1 , Activin RIB/ALK-4, GFR a2, Activin RHA, GFR a-3, Activin RUB, GFR a-4, ALK-1 , MIS Rll, ALK-7, Ret, BMPR-IA/ALK-3, TGF-bet a RI/ALK-5, BMPR- IB/ALK-6, TGF- ⁇ Rll, BMPR-II, TGF- ⁇ Rllb, Endoglin/CD 105 and TGF- ⁇ Rill.
  • TGF Transforming Growth Factor
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one Wnt-related molecule selected from the group consisting of Frizzled-1 , Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1 , Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzted-6, sFRP-4, Frizzled-7, MFRP, LRP 5, LRP 6, Wnt-1 , Wnt-8a, Wnt-3a, Wnt-IOb, Wnt-4, Wnt-11 , Wnt-5a, Wnt-9a and Wnt-7a.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one receptor ligand selected from the group consisting of 4-1 BB Ligand/TNFSF9, Lymphotoxin, APRIIJTNFSF 13,
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one tumor antigen selected from the group consisting of Squamous Cell Carcinoma Antigen 1 (SCCA-1 ), (PROTEIN T4-A), Squamous Cell Carcinoma Antigen 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B ; KIAA0049), MUCIN 1 (TUMOR-ASSOCIATED MUCIN;
  • EPISIALIN Tumor- Associated Epithelial Membrane Antigen
  • EMA Tumor- Associated Epithelial Membrane Antigen
  • H23AG Tumor-associated Epithelial Membrane Antigen
  • PUM PUM
  • Antigen DF3 CTCL tumor antigen sel-1 , CTCL tumor antigen sel4-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-l, CTCL tumor antigen se37-2, CTCL tumor antigen se57-l, CTCL tumor antigen se89-i, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1
  • cancer/testis antigen CT7 MAGE-B 1 ANTIGEN (MAGE-XP Antigen; DAM 10), MAGE-B2 Antigen (DAM6), MAGE-2 ANTIGEN, MAGE ⁇ 4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO- 45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1 , Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro-oncoiogical ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, Tumor- Associated Antigen CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell,
  • Serologically defined colon cancer antigen 1 Serologically defined breast cancer antigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16, Chromogranin A, parathyroid secretory protein 1 , DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one B cell target selected from the group consisting of CD10, CD19, CD20, CD21 , CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81 , CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDwl30, CD138 and CDwl50.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise at least one angiogenesis target is selected from the group consisting of Angiopoietin-1 , Angiopoietin-like 2, Angiopoietin-2,
  • Angiopoietin-like 3 Angiopoietin-3, Angiopoietin-like 7/CDT6, Angiopoietin-4, Tie-1 , Angiopoietin-like 1 , Tie-2, Angiogenin, iNOS, Coagulation Factor Ill/Tissue Factor, nNOS, CTGF/CCN2, NOV/CCN3, DANCE, OSM, EDG-1 , Plfr, EG- VEGF/PK1 , Proliferin, Endostatln, ROB 04, Erythropoietin, Thrombospondin-1 , Kininostatin, Thrombospondin-2, MFG-E8, Thrombospondin-4, Nitric Oxide, VG5Q, eNOS, EphAI, EphA5, EphA2, EphA6, EphA3, EphA7, EphA4, EphAS, EphBI, EphB
  • Ill/Tissue Factor E-Selectin, D6, P-Selectin, DC-SIGNR/CD299, SLAM,
  • EMMPRIN/CD 147 Tie-2, Endoglin/CD105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1 B, Erythropoietin R, TRAIL R1/TNFRSF10A, ESAM, TRAIL
  • R2/TNFRSF10B FABP5, VCAM-1 , ICAM-1/CD54, VEGF R2/Flk-1 , ICAM- 2/CD102, VEGF R3/Flt-4, IL-1 RI and VG5Q.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise a VSIG8 polypeptide according to the invention and at least one heterologous polypeptide and/or or binding moiety or VSIG8 polypeptides are linked to one another by an amino acid spacer.
  • VSIG8 polypeptides or fusion proteins according to the invention will a VSIG8 polypeptide and at least one heterologous polypeptide and/or or binding moiety or VSIG8 polypeptides are linked to one another by an amino acid spacer of sufficient length of amino acid residues so that the different moieties can successfully bind to their individual targets.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise 2-10 of any of the VSIG8 ECD polypeptides or fragments thereof disclosed herein.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise one or more VSIG8 polypeptide(s) and at least one heterologous polypeptide optionally intervened by a heterologous linker which optionally comprises a polypeptide that is not a fragment of a VSIG8
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise a linker which Is a peptide comprising 5-50 amino acid residues, more preferably 5-25 amino acid residues.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise a linker which comprises, consists essentially of, glycine, serine, and/or alanine residues.
  • VSIG8 polypeptides or fusion proteins according to the invention will comprise a linker which comprises 5-50, 5-25, 5-15, 4-14, 4-12, or more amino acid residues, e.g., which may include or consist of glycine, serine, and/or alanine residues.
  • VSIG8 fusion proteins optionally may contain a peptide or polypeptide linker domain that separates the VSIG8 polypeptide from the second
  • the linker domain contains the hinge region of an immunoglobulin.
  • the hinge region is derived from a human immunoglobulin. Suitable human immunoglobulins that the hinge can be derived from include IgG, IgD and IgA.
  • the hinge region is derived from human IgG. Amino acid sequences of immunoglobulin hinge regions and other domains are well known in the art.
  • the linker domain optionally contains a hinge region of an immunoglobulin as described above, and further includes one or more additional immunoglobulin domains.
  • Suitable peptide/polypeptide linker domains optionally include
  • Peptide linker sequences are at least 2 amino acids in length.
  • the peptide or polypeptide domains are flexible peptides or polypeptides.
  • a "flexible linker” herein refers to a peptide or polypeptide containing two or more amino acid residues joined by peptide bond(s) that provides increased rotational freedom for two polypeptides linked thereby than the two linked polypeptides would have in the absence of the flexible linker. Such rotational freedom allows two or more antigen binding sites joined by the flexible linker to each access target antigen(s) more efficiently.
  • Exemplary flexible peptides/polypeptides include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser, Ala- Ser, Gly-Gly- Gly-Ser, Gly4-Ser, (Gly4-Ser)2, (Gly4-Ser)3, (Gly4- Ser)4, [Gly4-Set]2 Gly-Ala-Gly-Ser- Gly4-Ser Gly- ⁇ Gly4-Ser ⁇ 2, Gly4-Ser-Gly, Gly-Ser- Gly2 and Gly-Ser-Gly2-Ser .
  • Additional flexible peptide/polypeptide sequences are well known in the art.
  • Other suitable peptide linker domains optionally include the TEV linker
  • ENLYFQG a linear epitope recognized by the Tobacco Etch Virus protease.
  • exemplary peptides/polypeptides include, but are not limited to,
  • VSIG8 fragments e.g., ECD fragments
  • VSIG8 fragments are linked to each other (multimers) and/or one or more VSIG8 fragments, e.g., ECD fragments, are linked to a heterologous polypeptide such as an immunoglobulin or fragment thereof, especially an immunoglobulin heavy chain or fragment thereof by a peptide linker, preferably a "flexible linker" sequence.
  • the linker sequence should allow effective positioning of the VSIG8 fragments and the heterologous polypeptide such as an immunoglobulin polypeptide or domains thereof to allow functional activity of both moieties and the domains thereof.
  • Successful presentation of the polypeptide fusion can modulate the activity of a cell either to induce or to inhibit T-cell proliferation, or to initiate or inhibit an immune response to a particular site. This can be determined in appropriate assays such as disclosed herein below, including the in vitro assays that includes sequential steps of culturing T cells to proliferate same, and contacting the T cells with a fusion polypeptide according to the invention or a cell expressing same and then evaluating whether the fusion polypeptide promotes or inhibits T cell proliferation.
  • polypeptide and the VSIG8 polypeptide is intended to mean that the domains of these moieties are positioned so that VSIG8 domains and heterologous polypeptide domains are capable of interacting with immune or other target cells, e.g., cancer or other VSIG8 expressing cells to initiate or inhibit an immune reaction, or to inhibit or stimulate cell development.
  • immune or other target cells e.g., cancer or other VSIG8 expressing cells to initiate or inhibit an immune reaction, or to inhibit or stimulate cell development.
  • the Fc domains preferably permits effective positioning of the F c domain and VS!GSdomains to allow functional activity of each domain.
  • the Fc domains are effectively positioned to allow proper fusion protein complex formation and/or interactions with F c receptors on immune cells or proteins of the complement system to stimulate F c -mediated effects including opsonization, ceil lysis, degranuiation of mast cells, basophils, and eosinophils, and other Fc receptor- dependent processes; activation of the complement pathway; and enhanced in vivo half-life of the fusion protein complex.
  • Linker sequences are discussed supra in connection with fusion proteins according to the invention.
  • Linker sequences can optionally be used to link two or more VSIG8 polypeptides of the biologically active polypeptide to generate a single-chain molecule with the desired functional activity.
  • the linker sequence comprises from about 5 to 20 amino acids, more preferably from about 7 or 8 to about 16 amino acids.
  • the linker sequence is preferably flexible so as not hold the VSIG8 polypeptide and moiety linked thereto, e.g., an effector molecule in a single undesired conformation.
  • the linker sequence can be used, e.g., to space the recognition site from the fused molecule.
  • the peptide linker sequence can be positioned between the biologically active VSIG8 polypeptide and the effector molecule, e.g., to chemically cross-link same and to provide molecular flexibility.
  • the linker in some embodiments will predominantly comprise amino acids with small side chains, such as glycine, alanine and serine, to provide for flexibility.
  • Preferably about 80 or 90 percent or greater of the linker sequence comprise glycine, alanine or serine residues, particularly glycine and serine residues.
  • Other suitable linker sequences include flexible linker designs that have been used successfully to join antibody variable regions together, see Whitlow, M.
  • the amino sequence of the linker should be capable of spanning a suitable distance from the C-terminal residue of the VSIG8 polypeptide to the N-terminal residue of the effector molecule.
  • Suitable linker sequences can be readily identified empirically. Additionally, suitable size and sequences of linker sequences also can be determined by known computer modeling techniques based on the predicted size and shape of the fusion polypeptide. Other linker sequences are discussed supra in connection with fusion proteins according to the invention.
  • a polypeptide as described herein comprises 2-20 VSIG8 ECD polypeptide fragments linked together.
  • the fragments are intervened by a heterologous linker which optionally comprises a polypeptide that is not a fragment of a VSIG8 polypeptide.
  • the linker is a peptide comprising 5-50 amino acid residues, more preferably 5-25 amino acid residues.
  • the linker comprises, consists essentially of, or consists of 4-12 glycine, serine, and/or alanine residues.
  • VSIG8 fusion proteins disclosed herein optionally contain a dimerization or multimerization or oligomerization domain that functions to dimerize, oligomerize or multimerize two or more fusion proteins, which may be the same or different (heteromultimers or homomultimers).
  • a VSIG8 fusion protein may be attached to another VSIG8 fusion protein or another moiety, e.g. another costimulatory fusion protein.
  • the domain that functions to dimerize or multimerize the fusion proteins can either be a separate domain, or alternatively can be contained within one of the other domains (VSIG8 polypeptide, second polypeptide, or peptide/polypeptide linker domain) of the fusion protein.
  • Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains.
  • dimerization or multimerization of fusion proteins can occur by chemical crosslinktng.
  • the dtmers or multimers that are formed can be
  • the second polypeptide "partner" in the VSIG8 fusion polypeptides may be comprised of one or more other proteins, protein fragments or peptides as described herein, including but not limited to any immunoglobulin (Ig) protein or portion thereof, preferably the Fc region, or a portion of a biologically or chemically active protein such as the papillomavirus E7 gene product, melanoma-associated antigen p97), and HIV env protein (gpl20).
  • the "partner” is optionally selected to provide a soluble dimer/multimer and/or for one or more other biological activities as described herein.
  • a "dimerization domain” is formed by the association of at least two amino acid residues or of at least two peptides or polypeptides (which may have the same, or different, amino acid sequences). The peptides or polypeptides may interact with each other through covalent and/or non- covalent associations).
  • Optional dimerization domains contain at least one cysteine that is capable of forming an intermolecular disulfide bond with a cysteine on the partner fusion protein.
  • the dimerization domain can contain one or more cysteine residues such that disulfide bond(s) can form between the partner fusion proteins.
  • dimerization domains contain one, two or three to about ten cysteine residues.
  • the dimerization domain is the hinge region of an immunoglobulin.
  • Additional exemplary dimerization domains can be any known in the art and include, but not limited to, coiled coils, acid patches, zinc fingers, calcium hands, a CHI-CL pair, an "interface" with an engineered “knob” and/or
  • protuberance as described in U. S. Patent No. 5,821 ,333, leucine zippers (e.g., from jun and/or fos) (U. S. Patent No. 5,932,448), and/or the yeast
  • WD40 Human et al., / Biol Cherrs., 273: 33489- 33494 (1998)) EH, Lim, "An isoleucine zipper, a receptor dimer pair (e.g., inteiieukin-8 receptor (IL-8R); and integrin heterodimers such as LFA-I and GPIIIb/llla), or the dimerization region(s) thereof, dimeric ligand polypeptides (e.g., nerve growth factor (NGF), neurotrophin-3 (NT-3), interieukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa, et al., J Biol.
  • NGF nerve growth factor
  • NT-3 neurotrophin-3
  • IL-8 vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor
  • polypeptide pairs can be identified by methods known in the art, including yeast two hybrid screens. Yeast two hybrid screens are described in U. S. Pat. Nos. 5,283,173 and 6,562,576. Affinities between a pair of interacting domains can be determined using methods known in the art, including as described in Katahira, et al., /. Biol Chem, 277, 9242-9246 (2002)).
  • a library of peptide sequences can be screened for heterod imerization , for example, using the methods described in WO 01/00814.
  • Useful methods for protein-protein interactions are also described in U. S. Patent No. 6,790,624.
  • a "multimerization domain” or “oligomerization domain” referred to herein is a domain that causes three or more peptides or polypeptides to interact with each other through covalent and/or non-covalent association(s).
  • Suitable multimerization or oligomerization domains include, but are not limited to, coiled- coil domains.
  • a coiled-coil is a peptide sequence with a contiguous pattern of mainly hydrophobic residues spaced 3 and 4 residues apart, usually in a sequence of seven amino acids (heptad repeat) or eleven amino acids (undecad repeat), which assembles (folds) to form a multimeric bundle of helices.
  • Coiled- coils with sequences including some irregular distribution of the 3 and 4 residues spacing are also contemplated.
  • Hydrophobic residues are in particular the hydrophobic amino acids Val, He, Leu, Met, Tyr, Phe and Trp.
  • hydrophobic means that at least 50% of the residues must be selected from the mentioned hydrophobic amino acids.
  • the coiled coil domain may be derived from laminin.
  • the heterotrimeric coiled coil protein laminin plays an important role in the formation of basement membranes. Hence, the multifunctional oligomeric structure is required for laminin function.
  • Coiled coil domains may also be derived from the thrombospondins in which three (TSP-I and TSP-2) or five (TSP-3, TSP-4 and TSP-5) chains are connected, or from COMP (COMPcc) (Guo, et at., EMBO J, 1998, 17: 5265-5272) which folds into a parallel five- stranded coiled coil (Malashkevich, et al., Science, 274: 761-765 (1996)).
  • COMP COMP
  • VASP vasodilator-stimulated phosphoprotein
  • CMP matrilin-1
  • viral fusion peptides soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein receptor (SNARE) complexes
  • leucine-rich repeats certain tRNA synthetases
  • VSIG8 polypeptides In another embodiment, VSIG8 polypeptides, fusion proteins, or
  • Antibodies suitable for use to multimerize VSIG8 polypeptides, fusion proteins, or fragments thereof include, but are not limited to, IgM antibodies and cross-linked, multivalent IgG, IgA, IgD, or IgE complexes.
  • Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains, including those described above. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. Fusion protein dimers can be homodimers or heterodimers. Fusion protein multimers can be homomultimers or
  • Fusion protein dimers as disclosed herein are of formula II: N-R1-R2-R3-C
  • N-R4-R5-R6-C or, alternatively, are of formula III: N-R1-R2-R3-C
  • formula II are defined as being in a parallel orientation and the fusion proteins of the dimer provided by formula III are defined as being in an antiparallel orientation.
  • Parallel and antiparallel dimers are also referred to as cis and trans dimers, respectively.
  • N and "C” represent the N- and C-termini of the fusion protein, respectively.
  • the fusion protein constituents "Rl”, “R2” and “R3” are as defined above with respect to formula I.
  • R4 is a VSIG8 polypeptide or a second polypeptide
  • R5" is an optional peptide/polypeptide linker domain
  • R6 is a VSIG8 polypeptide or a second polypeptide
  • R6 is a VSIG8 polypeptide when "R4" is a second polypeptide
  • R6 m is a second polypeptide when H R4" is a VSIG8 polypeptide.
  • “Rl” is a VSIG8 polypeptide
  • R4" is also a VSIG8 polypeptide
  • "R3" and “R8” are both second polypeptides.
  • Fusion protein dimers are defined as heterodimers when these conditions are not met for any reason.
  • heterodimers may contain domain orientations that meet these conditions (i. eorial for a dimer according to formula II, "Rl” and “R4" are both VSIG8 polypeptides, "R2" and "R5" are both
  • one polypeptide may contain a wild-type VSIG8 amino acid sequence while the other polypeptide may be a variant VSIG8 polypeptide.
  • An exemplary variant VSIG8 polypeptide is VSIG8, polypeptide that has been modified to have increased or decreased binding to a target cell, increased activity on immune cells, increased or decreased half-life or stability.
  • Dimers of fusion proteins that contain either a CHI or CL region of an immunoglobulin as part of the polypeptide linker domain preferably form heterodimers wherein one fusion protein of the dimer contains a CHI region and the other fusion protein of the dimer contains a CL region.
  • Fusion proteins can also be used to form multimers. As with dimers,
  • multimers may be parallel multimers, in which all fusion proteins of the multimer are aligned in the same orientation with respect to their N- and C- termini.
  • Multimers may be antiparallel multimers, in which the fusion proteins of the multimer are alternatively aligned in opposite orientations with respect to their Inland C-termini.
  • Multimers can be either homomultimers or heteromultimers.
  • the fusion protein is optionally produced in dimeric form; more preferably, the fusion is performed at the genetic level as described below, by joining polynucleotide sequences corresponding to the two (or more) proteins, portions of proteins and/or peptides, such that a joined or fused protein is produced by a cell according to the joined polynucleotide sequence.
  • the VSIG8 polypeptides and fusion proteins can contain a targeting
  • targeting domain to target the molecule to specific sites in the body.
  • Optional targeting domains target the molecule to areas of inflammation.
  • Exemplary targeting domains are antibodies, or antigen binding fragments thereof that are specific for inflamed tissue or to a proinflammatory cytokine including but not limited to IL17, IL-4, IL-6, IL-12, IL-21 , IL-22, IL-23, MIF, TNF-a, and TNF- ⁇ and combinations thereof.
  • the targeting domain may target the molecule to the CNS or may bind to VCAM-I on the vascular epithelium. Additional targeting domains can be peptide aptamers specific for a proinflammatory molecule.
  • the VSIG8 fusion protein can include a binding partner specific for a polypeptide displayed on the surface of an immune cell, for example a T cell.
  • the targeting domain specifically targets activated immune cells.
  • Optional immune cells that are targeted include ThO, Th1 , Th17, Th2 and Th22 T cells, other cells that secrete, or cause other cells to secrete inflammatory molecules including, but not limited to, IL- ⁇ , TNF-a, TGF- ⁇ , IFN-y, IL-17, IL-6, IL-23, IL-22, IL-21 , and MMPs, and Tregs.
  • a targeting domain for Tregs may bind specifically to CD25.
  • a protein according to the present invention is a linear molecule, it is possible to place various functional groups at various points on the linear molecule which are susceptible to or suitable for chemical modification.
  • Functional groups can be added to the termini of linear forms of the protein according to at least some embodiments of the invention, in some embodiments, the functional groups improve the activity of the protein with regard to one or more characteristics, including but not limited to, improvement in stability, penetration (through cellular membranes and/or tissue barriers), tissue
  • N-terminus of the compositions according to at least some embodiments of the invention will be termed as the N-terminus of the
  • compositions and the free C-terminal of the sequence will be considered as the C-terminus of the composition.
  • Non-limiting examples of suitable functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, (1991 ), the teachings of which are incorporated herein by reference.
  • Preferred protecting groups are those that facilitate transport of the active ingredient attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the active ingredient, these being an example for "a moiety for transport across cellular membranes”.
  • Hydroxyl protecting groups include esters, carbonates and carbamate protecting groups.
  • Amine protecting groups include alkoxy and aryloxy carbonyl groups, as described above for N- terminal protecting groups.
  • Carboxyiic acid protecting groups include aliphatic, benzylic and aryl esters, as described above for C-termina! protecting groups.
  • the carboxyiic acid group in the side chain of one or more glutamic acid or aspartic acid residue in a composition of the present invention is protected, preferably with a methyl, ethyl, benzyl or substituted benzyl ester, more preferably as a benzyl ester.
  • Non-limiting, illustrative examples of N-terminal protecting groups include acyl groups (-CO-R1 ) and alkoxy carbonyl or aryloxy carbonyl groups (-CO-0- R1 ), wherein Rl is an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or a substituted aromatic group.
  • acyl groups include but are not limited to acetyl, (ethyl ) ⁇ CO-, n-propyl-CO, iso-propyl-CO-, n- butyl-CO-, sec-butyl-CO-, t-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoyl phenyl-CO-, substituted phenyl-CO, benzyl-CO- and (substituted benzyl)- CO-.
  • alkoxy carbonyl and aryloxy carbonyl groups include CH3-0- CO-, (ethyl)-O-CO-, n-propyl-O-CO, iso-propyl-O-CO, n-butyl-O-CO, sec-butyt- O-CO, t-butyl-O-CO, phenyl-O- CO-, substituted phenyl-O-GO and benzyl-O- CO-, (substituted benzyl)- 0-CO-, Adamantan, naphtalen, myristoleyl, to!uen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane, norbomane, or Z-caproic.
  • one to four glycine residues can be present in the N-terminus of the molecule.
  • the carboxyl group at the C-terrninus of the compound can be protected, for example, by a group including but not limited to an amide (i. e., the hydroxyl group at the C-terminus is replaced with -NH 2, -NHR2 and -NR2R3) or ester (i. e., the hydroxyl group at the C-terminus is replaced with -OR2).
  • R2 and R3 are optionally independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a substituted aryl group.
  • R2 and R3 can optionally form a C4 to C8 heterocyclic ring with from about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur.
  • suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl.
  • C-terminal protecting groups include but are not limited to -NH 2l -NHCH3, -N(CH 3 ) 2 , - NH(ethyl), -N(ethyl) 2 , -N( methyl) (ethyl), -NH(benzyl), -N(C1-C4 alkyl)(benzyl), - NH(phenyl), -N(C1-C4 alkyl) (phenyl), -OCH3 -O-(ethyl), -O-(n-propyl), -0-(n- butyl), -0-(iso-piOpyl), -0-(sec- butyl), -0-(t-butyl ), -O-benzyi and -O-phenyl.
  • a "peptidomimetic organic moiety" can optionally be substituted for amino acid residues in the composition of this invention both as conservative and as non-conservative substitutions. These moieties are also termed “non-natural amino acids” and may optionally replace amino acid residues, amino acids or act as spacer groups within the peptides in lieu of deleted amino acids.
  • the peptidomimetic organic moieties optionally and preferably have steric, electronic or configurational properties similar to the replaced amino acid and such peptidomimetics are used to replace amino acids in the essential positions, and are considered conservative substitutions. However such similarities are not necessarily required. According to preferred embodiments of the present invention, one or more peptidomimetics are selected such that the composition at least substantially retains its physiological activity as compared to the native protein according to the present invention.
  • Peptidomimetics may optionally be used to inhibit degradation of the
  • peptides by enzymatic or other degradative processes.
  • the peptidomimetics can optionally and preferably be produced by organic synthetic techniques.
  • suitable peptidomimetics include D amino acids of the corresponding L amino acids, tetrazoi (Zabrocki et al., /. Am. Chem. Soc.
  • Exemplary, illustrative but non-limiting non-natural amino acids include ⁇ - amino acids ( ⁇ 3 and ⁇ 2), homo-amino acids, cyclic amino acids, aromatic amino acids, Pro and Pyr derivatives, 3-substituted Alanine derivatives, Glycine derivatives, ring-substituted Phe and Tyr Derivatives, linear core amino acids or diamirto acids. They are available from a variety of suppliers, such as Sigma- Aldrich (USA).
  • any part of a protein according to at least some embodiments of the invention may optionally be chemically modified, i. e. changed by addition of functional groups.
  • the side amino acid residues appearing in the native sequence may optionally be modified, although as described below alternatively other parts of the protein may optionally be modified, in addition to or in place of the side amino acid residues.
  • the modification may optionally be performed during synthesis of the molecule if a chemical synthetic process is followed, for example by adding a chemically modified amino acid.
  • chemical modification of an amino acid when it is already present in the molecule (“in situ" modification) is also possible.
  • Non- limiting exemplary types of modification include carboxymethylation, acyiation, phosphorylation, glycosylation or fatty acyiation.
  • Ether bonds can optionally be used to join the serine or threonine hydroxy I to the hydroxyl of a sugar.
  • Amide bonds can optionally be used to join the glutamate or aspartate carboxyl groups to an amino group on a sugar (Garg and Jeanloz, Advances in Carbohydrate Chemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang. Chem. Int. Ed. English 26:294-308 (1987)).
  • Acetal and ketal bonds can also optionally be formed between amino acids and carbohydrates.
  • Fatty acid acyl derivatives can optionally be made, for example, by acylation of a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry, Structure and Biology, Rivier and Marshal, eds., ESCOM Pub!., Leiden, 1078-1079 (1990)).
  • protein or peptide according to the present invention refers to a protein or peptide where at least one of its amino acid residues is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art.
  • Examples of the numerous known modifications typically include, but are not limited to: acetylation, acylation, amidation, ADP-ribosylation, glycosylate, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristoyiation, pegylation, prenylatlon, phosphorylation, ubiquitination, or any similar process.
  • cycloalkane moiety to a biological molecule, such as a protein, as described in PCT Application No. WO 2006/050262, hereby incorporated by reference as if fully set forth herein.
  • a biological molecule such as a protein
  • moieties are designed for use with biomolecules and may optionally be used to impart various properties to proteins.
  • any point on a protein may be modified.
  • pegylation of a glycosylation moiety on a protein may optionally be performed, as described in PCT Application No. WO 2006/050247, hereby incorporated by reference as if fully set forth herein.
  • One or more polyethylene glycol (PEG) groups may optionally be added to O-linked and/or N-linked glycosylation.
  • the PEG group may optionally be branched or linear.
  • any type of water-soluble polymer may be attached to a glycosylation site on a protein through a glycosyl linker.
  • Proteins according to at least some embodiments of the invention may be modified to have an altered glycosylation pattern (i. e., altered from the original or native glycosylation pattern).
  • altered means having one or more carbohydrate moieties deleted, and/or having at least one glycosylation site added to the original protein.
  • Glycosylation of proteins is typically either N- jinked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • O-linked glycosylation refers to the attachment of one of the sugars N- acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although S-hydroxyproline or 5-hydroxyiysine may also be used.
  • embodiments of the invention is conveniently accomplished by altering the amino acid sequence of the protein such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues in the sequence of the original protein (for O-linked glycosylation sites).
  • the protein's amino acid sequence may also be altered by introducing changes at the DNA level.
  • Another means of increasing the number of carbohydrate moieties on proteins is by chemical or enzymatic coupling of glycosides to the amino acid residues of the protein.
  • the sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxy! groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • Removal of any carbohydrate moieties present on proteins may be accomplished chemically or enzymatical!y.
  • Chemical deglycosylation requires exposure of the protein to trifluoromethanesutfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N- acetylglucosamine or N-acetylgalactosamine), leaving the amino acid sequence intact.
  • Enzymatic cleavage of carbohydrate moieties on proteins can be achieved by the use of a variety of endo- and exo-g!ycosidases as described by Thotakura et al., Meth. EnzymoL, 138: 350 (1987).
  • the invention further provides nucleic acids which encode an anti- VSIG8 antibody according to the invention, or a fragment or conjugate thereof.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCI banding, column
  • a nucleic acid according to at least some embodiments of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences, in a preferred embodiment, the nucleic acid is a cDNA molecule.
  • Nucleic acids according to at least some embodiments of the invention can be obtained using standard molecular biology techniques.
  • hybridomas e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PGR amplification or cDNA cloning techniques.
  • nucleic acid encoding the antibody can be recovered from the library.
  • VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a V L - or V H ⁇ encoding DMA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • "operatively linked" means that that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CHS).
  • CHI, CH2 and CHS heavy chain constant regions
  • the sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., el al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PGR amplification.
  • the heavy chain constant region can be an IgGI, lgG2, lgG3, lgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgGI, lgG2 or lgG4 constant region.
  • the Vn-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.
  • the isolated DNA encoding the V L region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V L - encoding DNA to another DNA molecule encoding the light chain constant region, CL-The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PGR amplification.
  • the light chain constant region can be a kappa ( ⁇ ) or lambda constant region, but most preferably is a ⁇ constant region.
  • a flexible linker e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
  • a flexible linker e.g., encoding the amino acid sequence (Gly4-Ser)3
  • Anti- VSIG8 monoclonal antibodies (mAbs) and antigen-binding fragments according to the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature
  • a preferred animal system for preparing hybridomas is the murine system.
  • Hybridoma production in the mouse is a very well-established procedure.
  • splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
  • Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared as described above.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.
  • the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Pat. No.
  • the murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter and U.S. Pat. Nos. 5,530,101 ;
  • the antibodies are human monoclonal antibodies.
  • Such human monoclonal antibodies directed against VSIG8 can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system.
  • mice referred to herein as the HuMAb MouseTM and KM MouseTM are coilectiveiy referred to herein as "human ig mice.”
  • the HuMAb MouseTM (Medarex Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy ⁇ and y and ⁇ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and ⁇ chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or ⁇ and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG ⁇ monoclonal
  • human antibodies according to at least some embodiments of the invention can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome.
  • KM miceTM a mouse that carries a human heavy chain transgene and a human light chain transchromosome.
  • KM miceTM are described in detail in PCT Publication WO 02/43478 to Ishida et al.
  • alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti- VSIG8 antibodies according to at least some embodiments of the invention.
  • an alternative transgenic system referred to as the Xenomouse
  • mice can be used; such mice are described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181 ; 6,114,598; 6, 150,584 and 6,162,963 to Kucheriapati et al.
  • human immunoglobulin genes are available in the art and can be used to raise anti-VSIG8 antibodies according to at least some embodiments of the invention.
  • mice carrying both a human heavy chain transchromosome and a human light chain transchromosome referred to as "TC mice” can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad Sci. USA 97:722- 727.
  • transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to raise anti-VSIG8 antibodies according to at least some embodiments of the invention.
  • Human monoclonal antibodies according to at least some embodiments of the invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. See for example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571 ,698 to Ladner et al.; U.S. Pat. Nos.
  • Human monoclonal antibodies according to at least some embodiments of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization.
  • SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization.
  • Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
  • human Ig mice are used to raise human anti-VSIG8 antibodies according to the invention, e.g., by immunizing such mice with a purified or enriched preparation of VSIG8 antigen and/or recombinant VSIG8 , or VSIG8 fusion protein, as described by Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwiid, D. et al. (1996) Nature Biotechnology 14: 845- 851 ; and PCT Publication WO 98/24884 and WO 01/14424.
  • the mice will be 6-16 weeks of age upon the first infusion.
  • a purified or recombinant preparation (5-50pg) of VSIG8 antigen can be used to immunize the human Ig mice intraperftoneally.
  • IP intra peritoneally
  • adjuvants other than Freund's are also found to be effective.
  • whole ceils in the absence of adjuvant are found to be highly immunogenic.
  • the immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds.
  • the plasma can be screened by ELISA (as described below), and mice with sufficient titers of anti-VSIG8 human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen.
  • mice are typically immunized for each antigen.
  • HCo7 and HCol2 strains are used.
  • both HCo7 and HCol2 transgene can be bred together into a single mouse having two different human heavy chain transgenes (HCo7/HCo 12).
  • the KM MouseTM strain can be used.
  • hybridomas producing a human monoclonal anti- VSIG8 antibody according to the invention may be generated using spienocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma ceil line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG.
  • Cells are plated at approximately 2X10 5 in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24 hours after the fusion). After approximately two weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies.
  • the antibody secreting hybridomas can be replated, screened again, and if still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.
  • selected hybridomas can be produced by:
  • Supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity.
  • the buffer solution can be exchanged into PBS, and the
  • concentration can be determined by OD280 using 1.43 extinction coefficient.
  • the monoclonal antibodies can be aliquoted and stored at -80 °C.
  • an anti-VS!G8 antibody according to the invention can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202).
  • DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PGR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • operatively linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or biunt end ligation if no restriction sites are present).
  • the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V H segment is operatively linked to the CH segments within the vector and the VL segment is operatively linked to the C L segment within the vector. Additionally or
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in- frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the binding specificity of an anti-VSIG8 antibody according to the invention is determined by known antibody binding assay techniques such as ELISA.
  • ELISA antibody binding assay techniques
  • microtiter plates are coated with a purified antigen, herein VSIG8 at 0.25pg/ml in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from -immunized mice) are added to each well and incubated for 1-2 hours at 37 °C.
  • the plates are washed with PBS/Tween and then incubated with secondary reagent (e.g., for human antibodies, a goat-anti-human IgG Fc- specific polyclonal reagent) conjugated to alkaline phosphatase for 1 hour at 37 °C. After washing, the plates are developed with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, mice which develop the highest titers will be used for fusions.
  • secondary reagent e.g., for human antibodies, a goat-anti-human IgG Fc- specific polyclonal reagent conjugated to alkaline phosphatase for 1 hour at 37 °C.
  • secondary reagent e.g., for human antibodies, a goat-anti-human IgG Fc- specific polyclonal reagent conjugated to alkaline phosphatase for 1 hour at 37 °C.
  • the plates are developed with pNPP substrate (1
  • hybridomas that show positive reactivity with VSIG8 immunogen Hybridomas that bind with high avidity to VSIG8 are subcloned and further characterized.
  • One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can be chosen for making a 5-10 vial cell bank stored at -140 °C, and for antibody purification.
  • hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification.
  • Supernatants can be filtered and concentrated before affinity chromatography with protein A- Sepharose (Pharmacia, Piscataway, N.J.).
  • Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity.
  • the buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient.
  • the monoclonal antibodies can be aliquoted and stored at -80 °C.
  • each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, 111.). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using VSIG8 coated-ELISA plates as described above. Biotinylated rnAb binding can be detected with a strep-avidin-alkaline phosphatase probe.
  • isotype ELISAs can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, wells of microtiter plates can be coated with A g/ml of anti-human immunoglobulin overnight at 4°C. After blocking with 1 % BSA, the plates are reacted with 1 mug /ml or less of test monoclonal antibodies or purified isotype controls, at ambient temperature for one to two hours. The wells can then be reacted with either human IgGi or human IgM- specific alkaline phosphatase-conjugated probes. Plates are developed and analyzed as described above.
  • Anti-VSIG8 human IgQs can be further tested for reactivity with VSIG8 antigen, respectively, by Western blotting. Briefly, VSIG8 antigen can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel
  • the present invention relates to an antigen- binding construct comprising a protein scaffold which is linked to one or more epitope-binding domains.
  • Such engineered protein scaffolds are usually obtained by designing a random library with mutagenesis focused at a loop region or at an otherwise permissible surface area and by selection of variants against a given target via phage display or related techniques.
  • the invention relates to alternative scaffolds including, but not limited to, anticalins, DARPins, Armadillo repeat proteins, protein A, lipocalins, fibronectin domain, ankyrin consensus repeat domain, thioredoxin, chemically constrained peptides and the like.
  • the invention relates to alternative scaffolds that are used as therapeutic agents for treatment of cancer, autoimmune, infectious diseases, sepsis, or for inhibiting an undesirable immune activation that follows gene therapy, as well as for in vivo diagnostics.
  • the invention further provides a pharmaceutical composition comprising an antigen-binding construct as described herein a pharmaceutically acceptable carrier.
  • the term 'Protein Scaffold' as used herein includes but is not limited to an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human or primate origin, or which may be an artificial chimera of human and primate constant regions.
  • Ig immunoglobulin
  • Such protein scaffolds may comprise antigen- binding sites in addition to the one or more constant regions, for example where the protein scaffold comprises a full IgG.
  • Such protein scaffolds will be capable of being linked to other protein domains, for example protein domains which have antigen- binding sites, for example epitope-binding domains or ScFv domains.
  • a "domain” is a folded protein structure which has tertiary structure
  • a "single antibody variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
  • immunoglobulin single variable domain refers to an antibody variable domain (VR, VRH, VL) that specifically binds an antigen or epitope independently of a different V region or domain.
  • An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other, different variable regions or variable domains where the other regions or domains are not required for antigen-binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains).
  • a "domain antibody” or “dAb” is the same as an "immunoglobulin single variable domain" which is capable of binding to an antigen as the term is used herein.
  • An immunoglobulin single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V-HH dAbs.
  • Camelid V-HH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains.
  • Such V- HH domains may be humanized according to standard techniques available in the art, and such domains are still considered to be "domain antibodies" according to the invention.
  • VH includes camelid V-HH domains.
  • NARV are another type of immunoglobulin single variable domain which was identified in cartilaginous fish including the nurse shark. These domains are also known as Novel Antigen Receptor variable region (commonly abbreviated to V (NAR) or NARV). See, e.g., Mol. Immunol. 44, 656-665 (2006) and
  • epitope-binding domain refers to a domain that specifically binds an antigen or epitope independently of a different V region or domain, this may be a domain antibody (dAb), for example a human, camelid or shark immunoglobulin single variable domain or it may be a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4
  • DARP ' in® peptide aptamer
  • Otype lectin domain Tetranectin
  • Lipocalins are a family of extracellular proteins which transport small
  • hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens.
  • Anticalins are between 160-180 amino acids in size, and are derived from iipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B 1 and US20070224633.
  • An affibody is a scaffold derived from Protein A of
  • Staphylococcus aureus which can be engineered to bind to antigen.
  • the domain consists of a three -helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP1641818A1 Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulphide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556 - 1561 (2005) and Expert Opinion on
  • Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop.
  • engineered transferrin scaffolds include the Trans- body. For further details see J. Biol. Chem 274, 24066-24073 (1999).
  • Designed Ankyrin Repeat Proteins are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton.
  • a single ankyrin repeat is a 33 residue motif consisting of two a helices;-P turn. They can be engineered to bind different target antigens by randomizing residues in the first a-helix and a ⁇ -tum of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.
  • Fibronectin is a scaffold which can be engineered to bind to antigen.
  • Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the P;-sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest.
  • Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a
  • Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges - examples of microproteins include KalataBI and conotoxin and knottins.
  • the microproteins have a loop which can be engineered to include up to 25 amino acids without affecting the overall fold of the microprotein.
  • engineered knottin domains see WO2008098796.
  • epitope binding domains include proteins which have been used as a scaffold to engineer different target antigen-binding properties include human ⁇ -crystallin and human ubiquitin (affilins), Kunitz type domains of human protease inhibitors, PDZ-domains of the Ras-binding protein AF-6, scorpion toxins (charybdo toxin), C-type lectin domain (tetranectins) are reviewed in Chapter 7 - Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15: 14-27 (2006) . Epitope binding domains of the present invention could be derived from any of these alternative protein domains.
  • the present invention encompasses conjugates of VSIG8 antigen for use in immune therapy comprising the VSIG8 antigen and soluble portions thereof including the ectodomain or portions or variants thereof.
  • the invention encompasses conjugates wherein the ECD of the VSIG8 antigen is attached to an immunoglobulin or fragment thereof.
  • the invention contemplates the use thereof for promoting or inhibiting VSIG8 antigen activities such as immune stimulation and the use thereof in treating transplant, autoimmune, and cancer indications described herein.
  • the present invention features antibody-drug conjugates (ADCs), used for example for treatment of cancer, consisting of an antibody (or antibody fragment such as a single-chain variable fragment (scFv) linked to a payload drug (often cytotoxic).
  • ADCs antibody-drug conjugates
  • the antibody causes the ADC to bind to the target cancer cells.
  • the ADC is then internalized by the cell and the drug is released into the cell. Because of the targeting, the side effects are lower and give a wider therapeutic window.
  • Hydrophilic linkers e.g., PEG4Mal
  • MDR multiple drug resistance
  • the present invention features immunoconjugates
  • a therapeutic agent such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • a therapeutic agent such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • cytotoxins Such conjugates are referred to herein as “immunoconjugates”.
  • Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins.”
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide.
  • Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g., vincristine and vinblastine.
  • therapeutic cytotoxins that can be conjugated to an antibody according to at least some embodiments of the invention include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof.
  • An example of a calicheamicin antibody conjugate is commercially available (MylotargTM Wyeth).
  • Cytotoxins can be conjugated to antibodies according to at least some embodiments of the invention using linker technology available in the art.
  • linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
  • a linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).
  • Antibodies of the present invention also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates.
  • radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine 131 , indium 111 , yttrium 90 and lutetium 177. Methods for preparing radioimmunconjugates are established in the art.
  • Radioimmunoconjugates are commercially available, including Zevalin®
  • the antibody or VSIG8 fusion proteins disclosed herein or conjugates according to at least some embodiments of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon- ⁇ ; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 ("IL- ⁇ ), interleukin-2 ("!L-2"), interieukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • an enzymatically active toxin, or active fragment thereof such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumor necrosis factor or interferon- ⁇
  • biological response modifiers such as, for example, lymphokines, interleukin- 1 ("IL- ⁇ ), interleukin-2 (“
  • the invention encompasses also a multispecific anti-VSIG8 antibody
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.
  • the present invention features bispecific molecules comprising an anti-VSIG8 antibody, or a fragment thereof, according to at least some embodiments of the invention.
  • An antibody according to at least some embodiments of the invention, or antigen-binding portions thereof can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • the antibody according to at least some embodiments of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein.
  • an antibody can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.
  • one of the binding specificities of the bispecific antibodies is for VSIG8 and the other is for any other antigen.
  • bispecific antibodies may bind to two different epitopes of VSIG8 .
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express VSIG8 .
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
  • a bispecific antibody according to at least some embodiments of the
  • Bispecific antibodies and bispecific antibody fragments (bsFab) according to at least some embodiments of the invention have at least one arm that specifically binds to a B-cell antigen or epitope and at least one other arm that specifically binds a targetable conjugate.
  • the invention encompasses also a fusion antibody protein, which is a recombinantly produced antigen-binding molecule in which two or more different single-chain antibody or antibody fragment segments with the same or different specificities are linked.
  • a fusion antibody protein which is a recombinantly produced antigen-binding molecule in which two or more different single-chain antibody or antibody fragment segments with the same or different specificities are linked.
  • a variety of bispecific fusion antibody proteins can be produced using molecular
  • the bispecific fusion antibody protein is monovalent, consisting of, for example, a sent with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen.
  • the bispecific fusion antibody protein is divalent, consisting of, for example, an IgG with two binding sites for one antigen and two scFv with two binding sites for a second antigen.
  • the invention further encompasses also engineered antibodies with three or more functional antigen-binding sites, including Octopus antibodies” (see, e.g. US 2006/0025576A1 ), and “Dual Acting FAb” or "DAP antibodies
  • the present invention includes bispecific molecules
  • the second target epitope is an Fc receptor, e.g., human FcyRI (CD64) or a human Fca receptor (CD89). Therefore, the invention includes bispecific molecules capable of binding both to FcyR, FcaR or FcsR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells
  • PMNs target cells expressing VSIG8
  • Fc receptor- mediated effector ceil activities such as phagocytosis of an VS1G8 expressing cells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.
  • bispecific molecule is multispecific, the molecule can further include a third binding specificity, in addition to an anti-Fc binding specificity.
  • the third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell.
  • EF anti-enhancement factor
  • the "anti-enhancement factor portion” can be an antibody, functional
  • the "anti-enhancement factor portion” can bind an Fc receptor or a target cell antigen.
  • the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind.
  • the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g., via CD2, CDS, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an increased immune response against the target cell).
  • the bispecific molecules comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv.
  • the antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Pat. No. 4,946,778, the contents of which are expressly incorporated by reference.
  • the binding specificity for an Fey receptor is provided by a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG).
  • IgG receptor refers to any of the eight ⁇ -chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms which are grouped into three Fey receptor classes: FcyRI (CD64), FcyRII(CD32), and FcyRIII (CD16).
  • FcyRI CD64
  • FcyRII CD32
  • FcyRIII CD16
  • the Fc y receptor is a human high affinity FcyRI.
  • the human FcyRI is a 72 kDa molecule, which shows high affinity for monomeric IgG .
  • the production and characterization of certain preferred anti-Fc y monoclonal antibodies are described by Fanger et al. in PCT Publication WO 88/00052 and in U.S. Pat.
  • H22 antibody producing cell line is deposited at the American Type Culture Collection under the designation
  • HA022CLI and has the accession no. CRL 11177.
  • IgA receptor is provided by an antibody that binds to a human IgA receptor, e.g., an Fc-a receptor (Fc aRI(CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA).
  • IgA receptor is intended to include the gene product of one a-gene (FcaRI) located on chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55 to 10 kDa. FcaRI (CD89) is constitutively expressed on
  • Fc aRI has medium affinity (Approximately 5X10 "7 M "1 ) for both IgAi and lgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in
  • FcaRI and FcyRI are preferred trigger receptors for use in the bispecific molecules according to at least some embodiments of the invention because they are (1 ) expressed primarily on immune effector cells, e.g., monocytes, PMNs, macrophages and dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000 per cell); (3) mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4) mediate enhanced antigen presentation of antigens, including self-antigens, targeted to them.
  • immune effector cells e.g., monocytes, PMNs, macrophages and dendritic cells
  • mediators of cytotoxic activities e.g., ADCC, phagocytosis
  • human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific molecules according to at least some embodiments of the invention are murine, chimeric and humanized monoclonal antibodies.
  • the bispecific molecules of the present invention can be prepared by
  • binding specificities e.g., the anti-FcR and anti- VSIG8 binding specificities
  • the binding specificity of each bispecific molecule can be generated separately and then conjugated to one another.
  • the binding specificities are proteins or peptides
  • a variety of coupling or cross-linking agents can be used for covalent conjugation.
  • cross-linking agents examples include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyld- ithio propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-l- carboxy!ate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
  • conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, II.).
  • binding moieties are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains.
  • the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.
  • both binding specificities can be encoded in the same vector and expressed and assembled In the same host ceil.
  • This method is particularly useful where the bispecific molecule is a mAbXmAb, mAbXFab, FabXF(ab')2 or ligandXFab fusion protein.
  • a bispecific molecule according to at least some embodiments of the invention can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants.
  • Bispecific molecules may comprise at least two single chain molecules. Methods for preparing bispecific molecules are described for example in U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No.
  • Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991 )), and "knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731 ,168).
  • Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1 ); controlled Fab-arm exchange (see Labrijn et a!., PNAS 110(13):5145-50 (2013)); cross- linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J.
  • ELISA enzyme-iinked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence-activated cell sorting
  • bioassay e.g., growth inhibition
  • Western Blot assay Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.
  • a labeled reagent e.g., an antibody
  • the FcR- antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes.
  • the complexes can be detected using any of a variety of other immunoassays.
  • the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein).
  • RIA radioimmunoassay
  • the radioactive isotope can be detected by such means as the use of a ⁇ counter or a scintillation counter or by autoradiography.
  • cancer immunotherapy is aimed to stimulate the patient's own immune system to eliminate cancer cells, providing long-lived tumor destruction.
  • tumors traditionally thought of as non-immunogenic can likely become immunogenic and respond to immunotherapy though co-administration of pro- immunogenic therapies designed to increase the patient's anti-tumor immune responses.
  • pro- immunogenic therapies designed to increase the patient's anti-tumor immune responses.
  • Potential priming agents are detailed herein below.
  • VSIG8 -specific antibodies, antibody fragments, conjugates and compositions comprising same are used for treatment of all types of cancer in cancer immunotherapy in combination therapy.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, which in this Example relates to treatment of cancer; however, also as described below, uses of antibodies and pharmaceutical compositions are also provided for treatment of infectious disease, sepsis, and/or autoimmune conditions, and/or for inhibiting an undesirable immune activation that follows gene therapy.
  • Those in need of treatment include those already with cancer as well as those in which the cancer is to be prevented.
  • the mammal to be treated herein may have been diagnosed as having the cancer or may be predisposed or susceptible to the cancer.
  • treating refers to preventing, delaying the onset of, curing, reversing, attenuating, alleviating, minimizing, suppressing, halting the deleterious effects or stabilizing of discernible symptoms of the above-described cancerous diseases, disorders or conditions. It also includes managing the cancer as described above.
  • manage it is meant reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, reducing the severity of such episodes, slowing/reducing cancer cell growth or proliferation, slowing progression of at least one symptom, amelioration of at least one measurable physical parameter and the like.
  • immunostimulatory anti-VSIG8 antibodies should promote T cell or NK or cytokine immunity against target cells, e.g., cancer, infected or pathogen cells and thereby treat cancer or infectious diseases by depleting the cells involved in the disease condition.
  • target cells e.g., cancer, infected or pathogen cells
  • immunoinhibitory anti-VSIG8 antibodies should reduce T cell or NK activity and/or or the secretion of proinflammatory cytokines which are involved in the disease pathology of some immune disease such as autoimmune, inflammatory or allergic conditions and thereby treat or ameliorate the disease pathology and tissue destruction that may be associated with such conditions (e.g., joint destruction associated with rheumatoid arthritis conditions).
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • the mammal is human.
  • the mammal is a human which is diagnosed with one of the disease, disorder or conditions described hereinabove, or alternatively one who is predisposed to at least one type of cancer.
  • terapéuticaally effective amount refers to an amount of agent according to the present invention that is effective to treat a disease or disorder in a mammal.
  • the therapeutic agents of the present invention can be provided to the subject alone, or as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.
  • An anti-VSIG8 antibody, a fragment, a conjugate thereof or VSIG8 fusion protein as herein described and/or a pharmaceutical composition comprising same, according to at least some embodiments of the present invention also can be administered in combination therapy, i.e., combined with other potentiating agents and/or other therapies.
  • the anti VSIG8 antibody or VSIG8 fusion proteins disclosed herein could be used in combination with any of the known in the art standard of care cancer treatment (as can be found, for example, in http://www.cancer.gov/cancertopics).
  • the combination therapy can include an anti VSIG8
  • antibody a fragment, a conjugate thereof and/or a pharmaceutical composition comprising same, combined with at least one other therapeutic or immune modulatory agent, other compounds or immunotherapies, or immuno stimulatory strategy as described herein.
  • therapeutic agents that can be used in combination with anti-VSIG8 antibodies are potentiating agents that enhance anti-tumor responses.
  • anti-VSIG8 antibody for cancer immunotherapy is used in combination with potentiating agents that are primarily geared to increase endogenous anti-tumor responses, such as Radiotherapy, Cryotherapy,
  • Targeted therapy potentiating anti-tumor immune responses, Anti- angiogenic therapy, Therapeutic agents targeting immunosuppressive cells such as Tregs and MDSCs, Immuno stimulatory antibodies, Cytokine therapy, Therapeutic cancer vaccines, Adoptive cell transfer.
  • immunosuppressive cells such as Tregs and MDSCs
  • Immuno stimulatory antibodies such as Cytokine therapy
  • Therapeutic cancer vaccines Adoptive cell transfer.
  • combination therapies that may potentiate anti-tumor responses through tumor cell death are radiotherapy, Cryotherapy, surgery, and hormone deprivation. Each of these cancer therapies creates a source of tumor antigen in the host.
  • anti- VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer are disclosed herein for cancer.
  • ABP amino- bisphosphonates
  • Targeted therapies can also stimulate tumor- specific immune response by inducing the immunogenic death of tumor ceils or by engaging immune effector mechanisms (Ga!luzzi et al, 2012, Nature Reviews - Drug discovery, Volume 11 , pages 215-233).
  • therapies used as agents for combination with anti VSIG8 antibodies for treatment of cancer are as described herein.
  • anti- VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer are disclosed herein for cancer.
  • immunotherapy is used in combination with Therapeutic agents targeting regulatory immunosuppressive cells such as regulatory T cells (Tregs) and myeloid derived suppressor ceils (MDSCs).
  • Tregs regulatory T cells
  • MDSCs myeloid derived suppressor ceils
  • a number of commonly used chemotherapeutics exert non-specific targeting of Tregs and reduce the number or the immunosuppressive capacity of Tregs or MDSCs (Facciabene A. et al 2012 Cancer Res; 72(9) 2162-71 ; Byrne WL et al 2011 , Cancer Res.
  • anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer immunotherapy is used in combination with drugs selected from but not limited to cyclophosphamide, gemcitabine, mitoxantrone, fludarabine, fludarabine, docetaxel, paciitaxel, thalidomide and thalidomide derivatives.
  • anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer immunotherapy is used in combination with novel Treg-specific targeting agents including: 1 ) depleting or killing antibodies that directly target Tregs through recognition of Treg cell surface receptors such as anti-CD25 mAbs daclizumab, basi!iximab or 2) ligand-directed toxins such as denileukin diftitox (Ontak) -a fusion protein of human IL-2 and diphtheria toxin, or LMB-2 - a fusion between an scFv against CD25 and Pseudomonas exotoxin and 3) antibodies targeting Treg ceil surface receptors such as CTLA4, PD-1 , OX40 and GITR or 4) antibodies, small molecules or fusion proteins targeting other NK receptors such as previously identified.
  • Treg-specific targeting agents including: 1 ) depleting or killing antibodies that directly target Tregs through recognition of Treg cell surface receptors such as anti-CD25
  • anti- VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer immunotherapy is used in combination with any of the options described below for disrupting Treg induction and/or function, including TLR ⁇ toil like receptors) agonists; agents that interfere with the adenosinergic pathway, such as ectonucieotidase inhibitors, or inhibitors of the A2A adenosine receptor, TGF- ⁇ inhibitors, such as fresolimumab, lerdelimumab, metelimumab, trabedersen, LY2157299, LY210976; blockade of Tregs recruitment to tumor tissues including chemokine receptor inhibitors, such as the CCR4/CCL2/CCL22 pathway.
  • TLR ⁇ toil like receptors TLR ⁇ toil like receptors
  • anti- VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer immunotherapy is used in combination with any of the options described below for inhibiting the immunosuppressive tumor microenvironment, including inhibitors of cytokines and enzymes which exert immunosuppressive activities, such as I DO (indoieamine-2,3-dioxygenase) inhibitors; inhibitors of antiinflammatory cytokines which promote an immunosuppressive
  • IL-10 IL-10, IL-35, IL-4 and IL-13
  • Bevacizumab® which reduces Tregs and favors the differentiation of DCs.
  • anti- VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer are disclosed herein for cancer.
  • immunotherapy is used in combination with any of the options described below for targeting MDSCs (myeloid-derived suppressive cells), including promoting their differentiation into mature myeloid cells that do not have suppressive functions by Vitamin D3, or Vitamin A metabolites, such as retinoic acid, all-trans retinoic acid (ATRA); inhibition of MDSCs suppressive activity by COX2 inhibitors, phosphodiesterase 5 inhibitors like sildenafil, ROS inhibitors such as nitroaspirin.
  • MDSCs myeloid-derived suppressive cells
  • ATRA all-trans retinoic acid
  • anti- VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer are disclosed herein for cancer.
  • immunotherapy is used in combination with immuno stimulatory antibodies or other agents which potentiate anti-tumor immune responses (Pardoll J Exp Med. 2012; 209(2): 201-209).
  • Immuno stimulatory antibodies promote anti-tumor immunity by directly modulating immune functions, i.e. blocking other inhibitory targets or enhancing immuno stimulatory proteins.
  • anti-VSIG8 antibody for cancer is used in combination with immuno stimulatory antibodies or other agents which potentiate anti-tumor immune responses.
  • immunotherapy is used in combination with antagonistic antibodies targeting immune checkpoints including anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-1 such as nivolumab BMS-936558/ MDX-1106/ONO- 4538, AMP224, CT-011 , MK-3475, anti-PDL-1 antagonists such as BMS- 936559/ MDX-1105, MEDI4736, RG-7446/MPDL3280A; Anti-LAG-3 such as IMP-321 ), anti-TIM-3, anti-BTLA, anti-B7-H4, anti-B7-H3, Anti- VISTA; Agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs such as BMS-663513 urelumab, PF-05082566; anti-OX40 mAb
  • Cytokines are molecular messengers that allow the cells of the immune system to communicate with one another to generate a coordinated, robust, but self -limited response to a target antigen. Cytokine-based therapies embody a direct attempt to stimulate the patient's own immune system to reject cancer. The growing interest over the past two decades in harnessing the immune system to eradicate cancer has been accompanied by heightened efforts to characterize cytokines and exploit their vast signaling networks to develop cancer treatments. Cytokines directly stimulate immune effector cells and stromal cells at the tumor site and enhance tumor cell recognition by cytotoxic effector cells.
  • cytokines have broad anti-tumor activity and this has been translated into a number of cytokine -based approaches for cancer therapy (Lee and Margolin 2011 , Cancers 3(4):3856-93 ).
  • a number of cytokines are in preclinical or clinical development as agents potentiating anti-tumor immune responses for cancer immunotherapy, including among others: IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21 , IL-23, IL-27, GM-CSF, IFNa (interferon a), ⁇ , and IFNy.
  • cytokines have been approved for therapy of cancer and many more are under development.
  • therapeutic efficacy is often hampered by severe side effects and poor pharmacokinetic properties.
  • a variety of strategies can be employed for the delivery of therapeutic cytokines and their localization to the tumor site, in order to improve their pharmacokinetics, as well as their efficacy and/or toxicity, including antibody- cytokine fusion molecules (immunocytokines), chemical conjugation to polyethylene glycol (PEGylation), transgenic expression of cytokines in autologous whole tumor cells, incorporation of cytokine genes into DNA vaccines, recombinant viral vectors to deliver cytokine genes, etc.
  • immunocytokines fusion of cytokines to tumor-specific antibodies or antibody fragments allows for targeted delivery and therefore improved efficacy and pharmacokinetics, and reduced side effects.
  • anti- VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer immunotherapy is used in combination with Cytokine therapy, involving the use of cytokines as agents potentiating anti-tumor immune responses, including cytokines such as IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21 , IL-23, IL-27, GM-CSF, IFNa (interferon a), IFNa-2b, IFNp, IFNy, and their different strategies for delivery, as described above.
  • cytokines such as IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21 , IL-23, IL-27, GM-CSF, IFNa (interferon a), IFNa-2b, IFNp, IFNy, and their different strategies for delivery, as described above.
  • Cancer vaccines are used to treat existing cancer (therapeutic) or prevent the development of cancer in certain high-risk individuals (prophylactic).
  • Therapeutic cancer vaccines allow for improved priming of T cells and improved antigen presentation, and can be used as therapeutic agents for potentiating anti-tumor immune responses (Mellman I. et al., 2011 , Nature, 480:22-29;
  • the tumor cell vaccine can be autologous, i.e. a patient's own tumor, or allogeneic which typically contain two or three established and characterized human tumor ceil lines of a given tumor type, such as the GVAX vaccine platforms.
  • Tumor antigen vaccines in which a tumor antigen (or a combination of a few tumor antigens), usually proteins or peptides, are administered to boost the immune system (possibly with an adjuvant and/or with immune modulators or attractants of dendritic cells such as GM-CSF).
  • the tumor antigens may be specific for a certain type of cancer, but they are not made for a specific patient.
  • Vector-based tumor antigen vaccines and DNA vaccines can be used as a way to provide a steady supply of antigens to stimulate an anti-tumor immune response.
  • Vectors encoding for tumor antigens are injected into the patient (possibly with proinflammatory or other attractants such as GM-CSF), taken up by cells in vivo to make the specific antigens, which would then provoke the desired immune response.
  • Vectors may be used to deliver more than one tumor antigen at a time, to increase the immune response.
  • recombinant virus, bacteria or yeast vectors should trigger their own immune responses, which may also enhance the overall immune response.
  • Oncolytic virus vaccines such as OncoVex/T-VEC, which involves the intratumoral injection of replication-conditional herpes simplex virus which preferentially infects cancer cells.
  • the virus which is also engineered to express GM-CSF, is able to replicate inside a cancer cell causing its lysis, releasing new viruses and an array of tumor antigens, and secreting GM-CSF in the process.
  • oncolytic virus vaccines enhance DCs function in the tumor microenvironment to stimulate anti-tumor immune responses.
  • DCs Dendritic cells
  • phagocytose tumor cells present tumor antigens to tumor specific T cells.
  • DCs are isolated from the cancer patient and primed for presenting tumor- specific T cells.
  • several methods can be used: DCs are loaded with tumor cells or lysates; DCs are loaded with fusion proteins or peptides of tumor antigens; coupling of tumor antigens to DC-targeting mAbs.
  • the DCs are treated in the presence of a stimulating factor (such as GM-CSF), activated and matured ex vivo, and then re-infused back into the patient in order provoke an immune response to the cancer cells.
  • a stimulating factor such as GM-CSF
  • Dendritic cells can also be primed in vivo by injection of patients with irradiated whole tumor cells engineered to secrete stimulating cytokines (such as GM-CSF). Similar approaches can be carried out with monocytes.
  • cytokines such as GM-CSF.
  • Sipuleucel-T Provenge
  • a therapeutic cancer vaccine which has been approved for treatment of advanced prostate cancer, is an example of a dendritic cell vaccine.
  • anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer immunotherapy is used in combination with Therapeutic cancer vaccines.
  • therapeutic cancer vaccines include Whole tumor cell vaccines, Tumor antigen vaccines, Vector-based vaccines, Oncolytic virus vaccines, Dendritic-cell vaccines, as described above.
  • adoptive cell transfer which involves the ex vivo identification and expansion of autologous naturally occurring tumor specific T cells, which are then adoptively transferred back into the cancer patient (Restifo et al, 2013, Cancer Immunol. lmmunother.62(4):727-36 (2013) Epub Dec 4 2012), Cells that are infused back into a patient after ex vivo expansion can traffic to the tumor and mediate its destruction.
  • hosts Prior to this adoptive transfer, hosts can be immunodepleted by irradiation and/or chemotherapy. The combination of lymphodepletion, adoptive cell transfer, and a T cell growth factor (such as IL-2), can lead to prolonged tumor eradication in tumor patients.
  • a more novel approach involves the ex vivo genetic modification of normal peripheral blood T cells to confer specificity for tumor-associated antigens.
  • clones of TCRs of T cells with particularly good anti-tumor responses can be inserted into viral expression vectors and used to infect autologous T cells from the patient to be treated.
  • CARs chimeric antigen receptors
  • CARs have antibody-like specificities and recognize MHC-nonrestricted structures on the surface of target cells (the extracellular target-binding module), grafted onto the TCR intracellular domains capable of activating T ceils (Restifo et al Cancer Immunol. lmmunother.62(4):727-36 (2013) Epub Dec 4 2012; and Shi et al, Nature 493: 111-115 2013.
  • anti- VSIG8 antibody or VSIG8 fusion proteins disclosed herein for cancer are disclosed herein for cancer.
  • immunotherapy is used in combination with Adoptive cell transfer to potentiate anti-tumor immune responses, including genetically modified T ceils, as described above.
  • compositions comprising same according to at least some embodiments of the present invention can be coadministered together with one or more other therapeutic agents, which acts in conjunction with or synergistically with the composition according to at least some embodiments of the present invention to treat or prevent the cancer.
  • the VSIG8 related therapeutic agents and the one or more other therapeutic agents can be administered in either order or simultaneously.
  • the other therapeutic agents are for example, a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent.
  • the composition can be linked to the agent (as an immunocomplex) or can be administered separately from the agent.
  • the composition can be administered before, after or concurrently with the agent or can be coadministered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation.
  • therapeutic agents include, among others, antineoplastic agents such as doxorubicin (Adriamycin), cisplatin bleomycin sulfate, canmustine, chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient.
  • Cisplatin is intravenously administered as a 100 mg/dose once every four weeks and
  • Adriamycin is intravenously administered as a 60-75 mg/ml dose once every 21 days.
  • Co-administration of the human anti-VSIG8 antibodies, or antigen-binding fragments and/or alternative scaffolds thereof, according to at least some embodiments of the present invention with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor ceils which would render them unreactive with the antibody.
  • the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fey or Fey receptors by, for example, treating the subject with a cytokine.
  • cytokines for administration during treatment with the multispecific molecule include of granulocyte colony- stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-y (IFN- ⁇ ), and tumor necrosis factor (T Fa or T FP).
  • Target-specific effector cells e.g., effector cells linked to compositions (e.g., human antibodies, multispecific and bispecific molecules) according to at ieast some embodiments of the present invention can also be used as compositions (e.g., human antibodies, multispecific and bispecific molecules) according to at ieast some embodiments of the present invention can also be used as compositions (e.g., human antibodies, multispecific and bispecific molecules) according to at ieast some embodiments of the present invention can also be used as
  • Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other IgG- or IgA-receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated.
  • the target- specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution.
  • the number of cells administered can be in the order of 10 " 8° to 10 - 9 * but will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization at the target cell, e.g., a tumor cell expressing VSIG8 proteins, and to effect cell killing e.g., by, e.g., phagocytosis. Routes of administration can also vary.
  • compositions e.g., human antibodies, multispecific and bispecific molecules
  • effector cells armed with these compositions can be used in conjunction with chemotherapy.
  • combination immunotherapy may be used to direct two
  • anti-VSIG8 antibodies linked to anti-Fc- ⁇ Rl or anti-CD3 may be used in conjunction with IgG- or IgA-receptor specific binding agents.
  • embodiments of the present invention can also be used to modulate FcyR or FcyR levels on effector cells, such as by capping and elimination of receptors on the ceil surface. Mixtures of anti-Fc receptors can also be used for this purpose.
  • compositions e.g., human antibodies, alternatively binds to human antibodies.
  • scaffolds multispecific and bispecific molecules and immunoconjugates according to at least some embodiments of the present invention which have complement binding sites, such as portions from IgGI, -2, or ⁇ 3 or IgM which bind complement, can also be used in the presence of complement, in one
  • ex vivo treatment of a population of cells comprising target cells with a binding agent according to at least some embodiments of the present invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement.
  • Phagocytosis of target cells coated with a binding agent according to at least some embodiments of the present invention can be improved by binding of complement proteins.
  • target cells coated with the compositions (e.g., human antibodies, multispecific and bispecific molecules) according to at least some embodiments of the present invention can also be lysed by complement.
  • the compositions according to at least some embodiments of the present invention do not activate complement.
  • compositions e.g., human antibodies, alternatively binds to human antibodies.
  • scaffolds multispecific and bispecific molecules and immunoconjugates according to at least some embodiments of the present invention can also be administered together with
  • compositions comprising human antibodies, multispecific or bispecific molecules and serum or complement.
  • compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules.
  • the human antibodies, multispecific or bispecific molecules according to at least some embodiments of the present invention and the complement or serum can be administered separately.
  • a "therapeutically effective dosage" of an anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in lifespan, disease remission, or a prevention or reduction of impairment or disability due to the disease affliction.
  • a "therapeutically effective dosage” preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • the ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors.
  • a therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine a therapeutically effective amount based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • the anti-VSIG8 antibodies can be used as neutralizing antibodies.
  • a Neutralizing antibody is an antibody that is capable of binding and neutralizing or inhibiting a specific antigen thereby inhibiting its biological effect, for example by blocking the receptors on the cell or the virus, inhibiting the binding of the virus to the host cell. NAbs will partially or completely abrogate the biological action of an agent by either blocking an important surface molecule needed for its activity or by interfering with the binding of the agent to its receptor on a target cell.
  • therapeutic agent is any one of the monoclonal and/or polyclonal antibodies, and/or antigen-binding fragments, and/or conjugates containing same, and/or alternative scaffolds, thereof comprising an antigen- binding site that binds specifically to any one of the VSIG8 polypeptides or an epitope thereof, adopted for treatment of cancer, as recited herein.
  • the therapeutic agents can be used to prevent pathologic inhibition of T cell activity, such as that directed against cancer cells.
  • the therapeutic agents can be used to inhibit T cell activation, as can be manifested for example by T cell proliferation and cytokine secretion.
  • a method of treating cancer as recited herein, and/or for promoting immune stimulation mediated by the VSIG8 polypeptide in a subject by administering to a subject in need thereof an effective amount of any one of the therapeutic agents and/or a pharmaceutical composition comprising any of the therapeutic agents and further comprising a pharmaceutically acceptable diluent or carrier.
  • a therapeutic agent or pharmaceutical composition according to at least some embodiments of the present invention may also be administered in conjunction with other compounds or immunotherapies.
  • the combination therapy can include a compound of the present invention combined with at least one other therapeutic or immune modulatory agent, or immuno stimulatory strategy, including, but not limited to, tumor vaccines, adoptive T cell therapy, Treg depletion, antibodies (e.g. bevacizumab, Erbitux), peptides, peptibodies, small molecules, chemotherapeutic agents such as cytotoxic and cytostatic agents (e.g.
  • paclitaxel paclitaxel
  • cisplatin vinorelbine
  • docetaxel gemcitabine
  • temozo!omide irinotecan, 5FU, carbop!atin
  • immunological modifiers such as interferons and interleukins, immuno stimulatory antibodies, growth hormones or other cytokines, folic acid, vitamins, minerals, aromatase inhibitors, RNAi, Histone Deacetylase Inhibitors, proteasome inhibitors, and so forth.
  • immune cells preferably T cells
  • T cells can be contacted in vivo or ex vivo with the therapeutic agents to modulate immune responses.
  • the T cells contacted with the therapeutic agents can be any cell which expresses the T cell receptor, including ⁇ / ⁇ and ⁇ / ⁇ T cell receptors.
  • T-cells include all cells which express CD3, including T-cell subsets which also express CD4 and CDS.
  • T-cells include both naive and memory cells and effector cells such as CTL.
  • T-cells also include cells such as Th1 , Tel, Th2, Th2, Th3, Thl7, Th22, Treg, and Trl cells.
  • T-cells also include NKT-cells and similar unique classes of the T-cell lineage.
  • VSIG8 blockade may also be combined with standard cancer treatments.
  • VSIG8 blockade may be effectively combined with chemotherapeutic regimes. In these instances, it may be possible to reduce the dose of chemotherapeutic reagent administered.
  • An example of such a combination is an anti ⁇ VS!G8 antibody or VSIG8 fusion proteins disclosed herein in combination with
  • Temsirolimus for the treatment of late stage renal cell cancer.
  • Another example of such a combination is an anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein in combination with interleukin-2 (IL-2) for the treatment of late stage renal cell cancer as well as combination with Ipilimumab or BMS-936558.
  • IL-2 interleukin-2
  • the scientific rationale behind the combined use of VSIG8 blockade and chemotherapy is that cell death, that is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway.
  • Other combination therapies that may result in synergy with VSIG8 blockade through cell death are radiotherapy, cryotherapy, surgery, and hormone deprivation.
  • angiogenesis leads to tumor cell death which may feed tumor antigen into host antigen presentation pathways.
  • VSIG8 blocking antibodies can also be used in combination with bispeciflc antibodies that target Fca or Fey receptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243).
  • Bispecific antibodies can be used to target two separate antigens.
  • anti-Fc receptor/anti- tumor antigen e.g., Her-2/neu
  • bispecific antibodies have been used to target macrophages to sites of tumor. This targeting may more effectively activate tumor specific responses.
  • the T cell arm of these responses would be augmented by the use of VSIG8 blockade.
  • antigen may be delivered directly to DCs by the use of bispecific antibodies which bind to tumor antigen and a dendritic cell specific cell surface marker.
  • TGF- ⁇ Kehrl, J. et at. (1986) J. Exp. Med. 163: 1037-1050
  • IL-10 Howard, M. & O'Garra, A. (1992)
  • Antibodies to each of these entities may be used in combination with anti-VSIG8 to counteract the effects of the immunosuppressive agent and favor tumor immune responses by the host.
  • anti-VSIG8 responsiveness can be used in combination with anti-VSIG8 .
  • These include molecules on the surface of dendritic cells which activate DC function and antigen presentation.
  • Anti-CD40 antibodies are able to substitute effectively for T ceil helper activity (Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in conjunction with VSIG8 antibodies (Ito, N. et al. (2000) Immunobiology 201 (5) 527-40).
  • Activating antibodies to T cell costimulatory molecules such as OX- 40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1 BB (Melero, I. et al.
  • Bone marrow transplantation is currently being used to treat a variety of tumors of hematopoietic origin. While graft versus host disease is a
  • VSIG8 blockade can be used to increase the
  • antibodies to VSIG8 can be combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), ceils, and cells transfected with genes encoding immune stimulating cytokines (He et al (2004) J. Immunol.
  • an immunogenic agent such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), ceils, and cells transfected with genes encoding immune stimulating cytokines (He et al (2004) J. Immunol.
  • Non-limiting examples of tumor vaccines that can be used include peptides of MUC1 for treatment of colon cancer, peptides of MUC- 1/CEA/TRICOM for the treatment of ovary cancer, or tumor ceils transfected to express the cytokine GM-CSF (discussed further below).
  • VSIG8 blockade is likely to be most effective when combined with a vaccination protocol.
  • Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61 , pp. 3023-3043 in DeVita, V. et al.
  • a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination
  • composition comprising same, as described herein, which function as VSIG8 stimulating therapeutic agents, may optionally be used for treating an immune system related disease.
  • the immune system related condition comprises an immune related condition, autoimmune diseases as recited herein, transplant rejection and graft versus host disease and/or for blocking or promoting immune
  • VSIG8 immune related diseases as recited herein and/or for immunotherapy (promoting or inhibiting immune stimulation).
  • the immune condition is selected from autoimmune disease, transplant rejection, or graft versus host disease.
  • the treatment is combined with another moiety useful for treating immune related condition.
  • treatment of multiple sclerosis using the agents according to at least some embodiments of the present invention may be combined with, for example, any known therapeutic agent or method for treating multiple sclerosis, optionally as described herein.
  • treatment of rheumatoid arthritis using the agents according to at least some embodiments of the present invention may be combined with, for example, any known therapeutic agent or method for treating rheumatoid arthritis, optionally as described herein.
  • treatment of IBD using the agents according to at least some embodiments of the present invention may be combined with, for example, any known therapeutic agent or method for treating IBD, optionally as described herein.
  • treatment of psoriasis using the agents according to at least some embodiments of the present invention may be combined with, for example, any known therapeutic agent or method for treating psoriasis, optionally as described herein.
  • treatment of type 1 diabetes using the agents according to at least some embodiments of the present invention may be combined with, for example, any known therapeutic agent or method for treating type Idiabetes, optionally as described herein.
  • treatment of uveitis using the agents according to at least some embodiments of the present invention may be combined with, for example, any known therapeutic agent or method for treating uveitis, optionally as described herein.
  • treatment for Sjogren's syndrome using the agents according to at least some embodiments of the present invention may be combined with, for example, any known therapeutic agent or method for treating for Sjogren's syndrome, optionally as described herein.
  • any known therapeutic agent or method for treating for systemic lupus erythematosus may be combined with, for example, any known therapeutic agent or method for treating for systemic lupus erythematosus, optionally as described herein.
  • aforementioned autoimmune or inflammatory conditions will be administered an immunoinhibitory anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein or antigen-binding fragment according to the invention, which antibody or VSIG8 fusion proteins disclosed herein mimics or agonizes at least one VSIG8 mediated effect on immunity, e.g., it suppresses cytotoxic T cells, or NK activity and/or the production of proinflammatory cytokines which are involved in the disease pathology, thereby preventing or ameliorating the disease symptoms and potentially resulting in prolonged disease remission, e.g., because of the induction of Tregs which elicit T cell tolerance or prolonged immunosuppression.
  • the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, according to at least some embodiments of the invention, may be administered as the sole active ingredient or together with other drugs in immunomodulating regimens or other anti-inflammatory agents e.g. for the treatment or prevention of alio- or xenograft acute or chronic rejection or inflammatory or autoimmune disorders, or to induce tolerance.
  • therapeutic agents may optionally be used for treating infectious disease.
  • T cell exhaustion can be reversed by blocking co-inhibitory pathways such as PD-1 or CTLA-4 (Rivas et a!., J Immunol. 2009 ; 183:4284-91 ; Golden-Mason et al., J Virol. 2009;83:9122-30; Hofmeyer et al., J Biomed Biotechnol. Vol 2011 , Art. ID 451694), thus allowing restoration of anti- viral immune function.
  • co-inhibitory pathways such as PD-1 or CTLA-4 (Rivas et a!., J Immunol. 2009 ; 183:4284-91 ; Golden-Mason et al., J Virol. 2009;83:9122-30; Hofmeyer et al., J Biomed Biotechnol. Vol 2011 , Art. ID 451694)
  • the therapeutic potential of co-inhibition blockade for treating viral infection was extensively studied by blocking the PD-I/PD-LI pathway, which was shown to be efficacious in several animal models of infection including acute and chronic simian immunodeficiency virus (SrV) infection in rhesus macaques (Valu et al., Nature 2009;458:206-210) and in mouse models of chronic viral infection, such as lymphocytic choriomeningitis virus (LCMV) (Barber et al., Nature.
  • SrV acute and chronic simian immunodeficiency virus
  • LCMV lymphocytic choriomeningitis virus
  • TMEV Theiler's murine encephalomyelitis virus
  • infection includes any disorder, disease and/or condition caused by presence and/or growth of pathogenic biological agent in an individual host organism.
  • the term “infection” comprises the disorder, disease and/or condition as above, exhibiting clinically evident illness (i.e., characteristic medical signs and/or symptoms of disease) and/or which is asymtomatic for much or all of it course.
  • the term “infection” also comprises disorder, disease and/or condition caused by persistence of foreign antigen that lead to exhaustion T cell phenotype characterized by impaired functionality which is manifested as reduced proliferation and cytokine
  • infectious disorder and/or disease and/or “infection”, further includes any of the below listed infectious disorders, diseases and/or conditions, caused by a bacterial infection, viral infection, fungal infection and /or parasite infection.
  • the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, can be administered in combination with one or more additional therapeutic agents used for treatment of bacterial infections, optionally as described herein.
  • the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, can be administered in combination with one or more additional therapeutic agents used for treatment of viral infections, optionally as described herein.
  • the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, can be administered in combination with one or more additional therapeutic agents used for treatment of fungal infections, optionally as described herein.
  • an immunostimulatory anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein or antigen- binding fragment will be administered an immunostimulatory anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein or antigen- binding fragment according to the invention, which antibody or VSIG8 fusion proteins disclosed herein antagonizes at least one VSIG8 mediated effect on immunity, e.g., its inhibitory effect on cytotoxic T cells or NK activity and/or its inhibitory effect on the production of proinflammatory cytokines, or inhibits the stimulatory effect of VSIG8 on Tregs thereby prompting the depletion or killing of the infected cells or the pathogen, and potentially resulting in disease remission based on enhanced killing of the pathogen or infected cells by the subject's immune cells.
  • aforementioned infectious conditions will be administered an immunostimulatory anti-VSIG8 antibody or VSIG8 fusion proteins disclosed herein or antigen- binding fragment according to the invention, which antibody or VSIG8 fusion proteins
  • VSIG8 antibodies, fragments, conjugates thereof and/or a pharmaceutical compositions as described herein, which function as VSIG8 blocking therapeutic agents, may optionally be used for treating sepsis.
  • Sepsis is a potentially life-threatening complication of an infection. Sepsis represents a complex clinical syndrome that develops when the initial host response against an infection becomes inappropriately amplified and
  • immunosuppression is increasingly recognized as the overriding immune dysfunction in these vulnerable patients.
  • the impaired pathogen clearance after primary infection and/or susceptibility to secondary infections contribute to the high rates of morbidity and mortality associated with sepsis.
  • a combination of the therapeutic agents and/or a pharmaceutical composition comprising same can be combined with standard of care or novel treatments for sepsis, with therapies that block the cytokine storm in the initial hyperinfiammatory phase of sepsis, and/or with therapies that have immunostimulatory effect in order to overcome the sepsis-induced immunosuppression phase.
  • Cefotaxime Cefotaxime
  • Ticarcillin and clavu!anate Timentin®
  • Piperacillin and tazobactam Zinc®
  • Imipenem and cilastatin Primarymaxin®
  • Meropenem Meropenem
  • Clindamycin Clindamycin
  • Vasopressors example Norepinephrine, Dopamine, Epinephrine,
  • Steroids example: Hydrocortisone, Dexamethasone, or Fludrocortisone, intravenous or otherwise Inotropic therapy: example Dobutamine for sepsis patients with myocardial dysfunction
  • rhAPC Recombinant human activated protein C
  • DrotAA drotrecogin alfa (activated)
  • ⁇ -blockers additionally reduce local and systemic inflammation.
  • sPLA2-IIA The Group IIA secretory phospholipase A2 (sPLA2-IIA), released during inflammation, is increased in severe sepsis, and plasma levels are inversely related to survival.
  • Phospholipid emulsion such as GR270773
  • Rationale Preclinical and ex vivo studies show that lipoproteins bind and neutralize endotoxin
  • anti-TNF- ⁇ antibody Rationale: Tumor necrosis factor-a (TNF-a) induces many of the pathophysiological signs and symptoms observed in sepsis anti-CD 14 antibody (such as IC14). Rationale: Upstream recognition molecules, like CD 14, play key roles in the pathogenesis. Bacterial cell wall components bind to CD 14 and co-receptors on myeloid cells, resulting in cellular activation and production of proinflammatory mediators. An anti-CD 14 monoclonal antibody (IC14) has been shown to decrease lipopolysaccharide-induced responses in animal and human models of endotoxemia.
  • TLRs Toll-like receptors
  • maeromolecules e.g., lipopolysaccharides, peptidoglycans
  • TLRs Toll-like receptors
  • TLR4 Various drugs targeting TLR4 expression and pathway have a therapeutic potential in sepsis (Wittebole et al 2010 Mediators of Inflammation Vol 10 Article ID 568396).
  • antibodies targeting TLR4 soluble TLR4, Statins (such as Rosuvastatin®, Simvastatin®), Ketamine, nicotinic analogues, eritoran (E5564), resatorvid (TAK242).
  • antagonists of other TLRs such as chbroquine, inhibition of TLR-2 with a neutralizing antibody (anti-TLR-2).
  • Lactoferrin is a glycoprotein with anti-infective and anti-inflammatory properties found in secretions and immune cells.
  • Talactoferrin alfa a recombinant form of human lactoferrin, has similar properties and plays an important role in maintaining the gastrointestinal mucosal barrier integrity. Talactoferrin showed efficacy in animal models of sepsis, and in clinical trials in patients with severe sepsis (Guntupal!i et al Crit Care Med. 2013;41(3):706-716).
  • MFG-E8 Milk fat globule EGF factor VIII - a bridging molecule between apoptotic cells and phagocytes, which promotes phagocytosis of apoptotic cells.
  • Agonists of the 'cholinergic anti -inflammatory pathway' such as nicotine and analogues. Rationale; Stimulating the vagus nerve reduces the production of cytokines, or immune system mediators, and blocks inflammation.
  • This nerve "circuitry”, called the “inflammatory reflex” is carried out through the specific action of acetylcholine, released from the nerve endings, on the a7 subunit of the nicotinic acetylcholine receptor (a7nAChR) expressed on macrophages, a mechanism termed 'the cholinergic antiinflammatory pathway' .
  • a7nAChR nicotinic acetylcholine receptor
  • vagus nerve stimulation or pharmacologic a7 agonists prevents tissue injury in multiple models of systemic inflammation, shock, and sepsis (Matsuda et al 2012 J Nippon Med Sch.79:4-18; Huston 2012 Surg. Infect. 13: 187-193).
  • MSCs Mesenchymal stem cells
  • neoangiogenesis and exhibit direct antimicrobial activity. These effects are associated with reduced organ dysfunction and improved survival in sepsis animal models, which have provided evidence that MSCs may be useful therapeutic adjuncts (Wannemuehler et al 2012 J. Surg. Res. 173: 113-26).
  • Immunostimulatory antibodies promote immune responses by directly modulating immune functions, i.e. blocking other inhibitory proteins or by enhancing costimulatory proteins.
  • Experimental models of sepsis have shown that immuno stimulation by antibody blockade of inhibitory proteins, such as PD- 1 , PDL-1 or CTLA-4 improved survival in sepsis (Brahmamdam et a! 2010 J. Leukoc. Biol.
  • Immunostimulatory antibodies include: 1 ) Antagonistic antibodies targeting inhibitory immune checkpoints include anti-CTLA4 mAbs (such as ipilimumab, tremelimumab), Anti-PD-1 (such as nivolumab BMS- 936558/ MDX-1106/ONO-4538, AMP224, CT-011 , lambrozitumab MK-3475), Anti-PDL-1 antagonists (such as BMS-936559/ MDX-1105, MEDI4736, RG- 7446/MPDL3280A); Anti-LAG-3 such as IMP-321 ), Anti-TIM-3, Anti-BTLA, Anti- B7-H4, Anti-B7-H3, anti- VISTA. 2) Agonistic antibodies enhancing
  • immunostimulatory proteins include Anti-CD40 mAbs (such as CP-870,893, lucatumumab, dacetuzumab), Anti-CD137 mAbs (such as BMS-663513 urelumab, PF-05082566), Anti-OX40 mAbs (such as Anti-OX40), Anti-GITR mAbs (such as TRX518), Anti-CD27 mAbs (such as CDX-1127), and Anti-ICOS mAbs.
  • Anti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab
  • Anti-CD137 mAbs such as BMS-663513 urelumab, PF-05082566
  • Anti-OX40 mAbs such as Anti-OX40
  • Anti-GITR mAbs such as TRX5128
  • Anti-CD27 mAbs such as CDX-1127
  • Cytokines which directly stimulate immune effector cells and enhance immune responses can be used in combination with anti-GEN antibody for sepsis therapy: IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21 , IL-23, IL-27, GM- CSF, IFNa (interferon a), ⁇ , IFNy.
  • Cytokine-based therapies embody a direct attempt to stimulate the patient's own immune system.
  • IFNy and granulocyte-macrophage colony- stimulating factor restore immune competence of ex vivo stimulated leukocytes of patients with sepsis (Mouktaroudi et a! Crit Care. 2010; 14: P17; Leentjens et ai Am J Respir Crit Care Med Vol 186, pp 838-845, 2012).

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Abstract

Selon la présente invention, le récepteur de VISTA est identifié (VSIG8) ainsi que l'utilisation de ce récepteur dans l'identification ou la synthèse de composés agonistes ou antagonistes, de préférence des anticorps, des polypeptides et des protéines de fusion qui ont un effet agoniste et/ou antagoniste sur les effets de VSIG8 et/ou VISTA et/ou l'interaction de liaison VSIG8/VISTA. Ces antagonistes peuvent être utilisés pour supprimer les effets suppresseurs de VISTA sur l'immunité à lymphocytes T, et plus particulièrement utilisés dans le traitement du cancer, ou d'une maladie infectieuse. Ces composés agonistes peuvent être utilisés pour potentialiser ou amplifier les effets suppresseurs de VISTA sur l'immunité à lymphocytes T et supprimer ainsi l'immunité à lymphocytes T, par exemple dans le traitement d'une auto-immunité, d'une allergie ou d'affections inflammatoires. L'invention concerne en outre des essais de criblage pour identifier ces composés agonistes et antagonistes.
PCT/US2015/064146 2014-12-05 2015-12-05 Identification de vsig8 en tant que récepteur putatif de vista et son utilisation pour produire des modulateurs de vista/vsig8 WO2016090347A1 (fr)

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CN201580075472.6A CN107405398A (zh) 2014-12-05 2015-12-05 鉴定vsig8作为推定vista受体及其用以产生vista/vsig8激动剂和拮抗剂的用途
MX2017007136A MX2017007136A (es) 2014-12-05 2015-12-05 Identificacion de vsig8 como el receptor vista putativo y su uso para producir moduladores vista/vsig8.
CA2969730A CA2969730A1 (fr) 2014-12-05 2015-12-05 Identification de vsig8 en tant que recepteur putatif de vista et son utilisation pour produire des modulateurs de vista/vsig8
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AU2015357463A AU2015357463B2 (en) 2014-12-05 2015-12-05 Identification of VSIG8 as the putative vista receptor and its use thereof to produce vista/VSIG8 modulators
US14/960,973 US10370455B2 (en) 2014-12-05 2015-12-07 Identification of VSIG8 as the putative VISTA receptor (V-R) and use thereof to produce VISTA/VSIG8 agonists and antagonists
IL252620A IL252620A0 (en) 2014-12-05 2017-06-01 Identification of vsig8 as a potential vista receptor and its use to prepare vista/vsig8 modulators
US16/531,968 US20200199254A1 (en) 2014-12-05 2019-08-05 Identification of vsig8 as the putative vista receptor (v-r) and use thereof to produce vista/vsig8 agonists and antagonists
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